Human metabotropic glutamate receptor subtypes (hmR4, hmR6, hmR7) and related DNA compounds

ABSTRACT

The present invention relates to human metabotropic glutamate receptor (hmGluR) proteins, isolated nucleic acids coding therefor, host cells producing the proteins of the invention, methods for the preparation of such proteins, nucleic acids and host cells, and uses thereof.

The present invention relates to human metabotropic glutamate receptor(hmGluR) proteins, isolated nucleic acids coding therefor, host cellsproducing the proteins of the invention, methods for the preparation ofsuch proteins, nucleic acids and host cells, and uses thereof.Furthermore, the invention provides antibodies directed against thehmGluR proteins of the invention.

Metabotropic glutamate receptors (hmGluR) belong to the class ofG-protein (guanine nucleotide binding protein) coupled receptors whichupon binding of a glutamatergic ligand may transduce an extracellularsignal via an intracellular second messenger system such as calciumions, a cyclic nucleotide, diacylglycerol and inositol1,4,5-triphosphate into a physiological response. Possessing sevenputative transmembrane spanning segments, preceded by a largeextracellular amino-terminal domain and followed by a largecarboxy-terminal domain metabotropic glutamate receptors arecharacterized by a common structure. Based on the degree of sequenceidentity at the amino acid level the class of mGluR can be divided intodifferent subfamilies comprising individual receptor subtypes(Nakanishi, Science 258, 597-603 (1992)). Each mGluR subtype is encodedby a unique gene. Regarding the homology of an individual mGluR subtypeto another subtype of a different subfamily, the amino acid sequencesare less than about 50% identical. Within a subfamily the degree ofsequence identity is generally less than about 70%. Thus a particularsubtype may be characterized by its amino acid sequence homology toanother mGluR subtype, especially a subtype of the same mammalianspecies. Furthermore, a particular subtype may be characterized by itsregion and tissue distribution, its cellular and subcellular expressionpattern or by its distinct physiological profile, e.g. by itselectrophysiological and pharmacological properties.

The amino acid L-glutamate being the major excitatory neurotransmitter,glutamatergic systems are presumed to play an important role in numerousneuronal processes including fast excitatory synaptic transmission,regulation of neurotransmitter releases, long-term potentation, learningand memory, developmental synaptic plasticity, hypoxic-ischemic damageand neuronal cell death, epileptiform seizures, as well as thepathogenesis of several neurodegenerative disorders. Up to today, noinformation is available on human metabotropic glutamate receptor(hmGluR) subtypes, e.g. on their amino acid sequence or tissuedistribution. This lack of knowledge particularly hampers the search forhuman therapeutic agents capable of specifically influencing anydisorder attributable to a defect in the glutamatergic system. In viewof the potential physiological and pathological significance ofmetabotropic glutamate receptors, there is a need for human receptorsubtypes and cells producing such subtypes in amounts sufficient forelucidating the electrophysiological and pharmacological properties ofthese proteins. For example, drug screening assays require purifiedhuman receptor proteins in an active form, which have not yet beenattainable.

It is an object of the present invention to fulfill this need, namely toprovide distinct hmGluR subtypes, nucleic acids coding therefor and hostcells producing such subtypes. In particular, the present inventiondiscloses the hmGluR subfamily comprising the subtype designatedhmGluR4, and the individual proteins of said subfamily. In thefollowing, said subfamily will be referred to as the hmGluR4 subfamily.Contrary to other hmGluR subtypes the members of this subfamily arepotently activated by L-2-amino-4-phosphobutyric acid (AP4) and, whenexpressed e.g. in Chinese hamster ovary (CHO) cells or baby hamsterkidney (BHK) cells, negatively coupled to adenylate cyclase via Gprotein. Using a system comprising a recombinant hmGluR subtype of theinvention in screening for hmGluR reactive drugs offers (among others)the possibilities of attaining a greater number of receptors per cellgiving greater yield of reagent and a higher signal to noise ratio inassays as well as increased receptor subtype specificity (potentiallyresulting in greater biological and disease specificity).

More specifically, the present invention relates to a hmGluR subtypecharacterized in that its amino acid sequence is more than about 65%identical to the sequence of the hmGluR4 subtype having the amino acidsequence depicted in SEQ ID NO:2.

According to the invention the expression “hmGluR subtype” refers to apurified protein which belongs to the class of G protein-coupledreceptors and which upon binding of a glutamatergic ligand transduces anextracellular signal via an intracellular second messenger system. Insuch case, a subtype of the invention is characterized in that itmodifies the level of a cyclic nucleotide (cAMP, cGMP). Alternatively,signal transduction may occur via direct interaction of the G proteincoupled to a receptor subtype of the invention with another membraneprotein, such as an ion channel or another receptor. A receptor subtypeof the invention is believed to be encoded by a distinct gene which doesnot encode another metabotropic glutamate receptor subtype. A particularsubtype of the invention may be characterized by its distinctphysiological profile, preferably by its signal transduction andpharmacological properties. Pharmacological properties are e.g. theselectivity for agonists and antagonist responses.

As defined herein, a glutamatergic ligand is e.g. L-glutamate or anothercompound interacting with, and particularly binding to, a hmGluR subtypein a glutamate like manner, such as ACPD(1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid), an ACPD-like ligand,e.g. QUIS (quisqualate), AP4, and the like. Other ligands, e.g.(R,S)-α-methylcarboxyphenylglycine (MCPG) or α-methyl-L-AP4, mayinteract with a receptor of the invention in such a way that binding ofglutamatergic ligand is prevented.

As used hereinbefore or hereinafter, the terms “purified” or “isolated”are intended to refer to a molecule of the invention in an enriched orpure form obtainable from a natural source or by means of geneticengineering. The purified proteins, DNAs and RNAs of the invention maybe useful in ways that the proteins, DNAs and RNAs as they naturallyoccur are not, such as identification of compounds selectivelymodulating the expression or the activity of a hmGluR of the invention.

Purified hmGluR of the invention means a member of the hmGluR4 subfamilywhich has been identified and is free of one or more components of itsnatural environment. Purified hmGluR includes purified hmGluR of theinvention in recombinant cell culture. The enriched form of a subtype ofthe invention refers to a preparation containing said subtype in aconcentration higher than natural, e.g. a cellular membrane fractioncomprising said subtype. If said subtype is in a pure form it issubstantially free from other macromolecules, particularly fromnaturally occurring proteinaceous contaminations. If desired, thesubtype of the invention may be solubilized. A preferred purified hmGluRsubtype of the invention is a recombinant protein. Preferably, thesubtype of the invention is in an active state meaning that it has bothligand binding and signal transduction activity. Receptor activity ismeasured according to methods known in the art, e.g. using a bindingassay or a functional assay, e.g. an assay as described below.

Preferred hmGluR subtypes of the hmGluR4 subfamily are subtypes hmGluR4,hmGluR7 and hmGluR6. A particularly preferred hmGluR4 subtype is theprotein having the amino acid sequence set forth in SEQ ID NO:2. AhmGluR7-type protein may comprise a polypeptide selected from the groupconsisting of the polypeptides having the amino acid sequences depictedin SEQ ID NOs: 4, 6, 8 and 10, respectively. Such hmGluR7 subtype ispreferred. Particularly preferred are the hmGluR7 subtypes having theamino acid sequences set forth in SEQ ID NOs: 12 and 14, respectively. Apreferred hmGluR6-type protein comprises a polypeptide having the aminoacid sequence depicted in SEQ ID NO:16.

The invention is further intended to include variants of the receptorsubtypes of the invention. For example, a variant of a hmGluR subtype ofthe invention is a functional or immunological equivalent of saidsubtype. A functional equivalent is a protein, particularly a humanprotein, displaying a physiological profile essentially identical to theprofile characteristic of said particular subtype. The physiologicalprofile in vitro and in vivo includes receptor effector function,electrophysiological and pharmacological properties, e.g. selectiveinteraction with agonists or antagonists. Exemplary functionalequivalents may be splice variants encoded by mRNA generated byalternative splicing of a primary transcript, amino acid mutants andglycosylation variants. An immunological equivalent of a particularhmGluR subtype is a protein or peptide capable of generating antibodiesspecific for said subtype. Portions of the extracellular domain of thereceptor, e.g. peptides consisting of at least 6 to 8 amino acids,particularly 20 amino acids, are considered particularly usefulimmunological equivalents.

Further variants included herein are membrane-bound and solublefragments and covalent or aggregative conjugates with other chemicalmoieties, these variants displaying one or more receptor functions, suchas ligand binding or signal transduction. Exemplary fragments of hmGluRsubtypes of the invention are the polypeptides having the amino acidsequences set forth in SEQ ID NOs: 4, 6, 8, 10 and 16, respectively. Thefragments of the invention are obtainable from a natural source, bychemical synthesis or by recombinant techniques. Due to their capabilityof competing with the endogenous counterpart of a hmGluR subtype of theinvention for its endogenous ligand, fragments, or derivatives thereof,comprising the ligand binding domain are envisaged as therapeuticagents.

Covalent derivatives include for example aliphatic esters or amides of areceptor carboxyl group, O-acyl derivatives of hydroxyl group containingresidues and N-acyl derivatives of amino group containing residues. Suchderivatives can be prepared by linkage of functionalities to reactablegroups which are found in the side chains and at the N- and C-terminusof the receptor protein. The protein of the invention can also belabeled with a detectable group, for example radiolabeled, covalentlybound to rare earth chelates or conjugated to a fluorescent moiety.

Further derivatives are covalent conjugates of a protein of theinvention with another protein or peptide (fusion proteins). Examplesare fusion proteins comprising different portions of different glutamatereceptors. Such fusion proteins may be useful for changing the couplingto G-proteins and/or improving the sensitivity of a functional assay.For example, in such fusion proteins or chimeric receptors, theintracellular domains of a subtype of the invention may be replaced withthe corresponding domains of another mGluR subtype, particularly anotherhmGluR subtype, e.g. a hmGLuR subtype belonging to another subfamily.Particularly suitable for the construction of such a chimeric receptorare the intracellular domains of a receptor which activates thephospholipase C/Ca²⁺ signaling pathway, e.g. mGluR1 (Masu et al., Nature349, 760-765) or mGluR5. An intracellular domain suitable for such anexchange is e.g. the second intracellular loop, also referred to as i2(Pin et al., EMBO J. 13, 342-348 (1994)). Thus it is possible to analyzethe interaction of a test compound with a ligand binding domain of areceptor of the invention using an assay for calcium ions. The chimericreceptor according to the invention can be synthesized by recombinanttechniques or agents known in the art as being suitable for crosslinkingproteins.

Aggregative derivatives are e.g. adsorption complexes with cellmembranes.

In another embodiment, the present invention relates to a composition ofmatter comprising a hmGluR subtype of the invention.

The proteins of the invention are useful e.g. as immunogens, in drugscreening assays, as reagents for immunoassays and in purificationmethods, such as for affinity purification of a binding ligand.

A protein of the invention is obtainable from a natural source, e.g. byisolation from brain tissue, by chemical synthesis or by recombinanttechniques.

The invention further provides a method for preparing a hmGluR subtypeof the invention characterized in that suitable host cells producing areceptor subtype of the invention are multiplied in vitro or in vivo.Preferably, the host cells are transformed (transfected) with a hybridvector comprising an expression cassette comprising a promoter and a DNAsequence coding for said subtype which DNA is controlled by saidpromoter. Subsequently, the hmGluR subtype of the invention may berecovered. Recovery comprises e.g. isolating the subtype of theinvention from the host cells or isolating the host cells comprising thesubtype, e.g. from the culture broth. Particularly preferred is a methodfor preparation of a functionally active receptor.

HmGluR muteins may be produced from a DNA encoding a hmGluR protein ofthe invention which DNA has been subjected to in vitro mutagenesisresulting e.g. in an addition, exchange and/or deletion of one or moreamino acids. For example, substitutional, deletional and insertionalvariants of a hmGluR subtype of the invention are prepared byrecombinant methods and screened for immuno-crossreactivity with thenative forms of the hmGluR.

A protein of the invention may also be derivatized in vitro according toconventional methods known in the art.

Suitable host cells include eukaryotic cells, e.g. animal cells, plantcells and fungi, and prokaryotic cells, such as gram-positive andgram-negative bacteria, e.g. E. coli. Preferred eukaryotic host cellsare of amphibian or mammalian origin.

As used herein, in vitro means ex vivo, thus including e.g. cell cultureand tissue culture conditions.

This invention further covers a nucleic acid (DNA, RNA) comprising apurified, preferably recombinant, nucleic acid (DNA, RNA) coding for asubtype of the invention, or a fragment of such a nucleic acid. Inaddition to being useful for the production of the above recombinanthmGluR proteins, these nucleic acid are useful as probes, thus readilyenabling those skilled in the art to identify and/or isolate nucleicacid encoding a hmGluR protein of the invention. The nucleic acid may beunlabeled or labeled with a detectable moiety. Furthermore, nucleic acidaccording to the invention is useful e.g. in a method for determiningthe presence of hmGluR, said method comprising hybridizing the DNA (orRNA) encoding (or complementary to) hmGluR to test sample nucleic acidand to determine the presence of hmGluR.

Purified hmGluR encoding nucleic acid of the invention includes nucleicacid that is free from at least one contaminant nucleic acid with whichit is ordinarily associated in the natural source of hmGluR nucleicacid. Purified nucleic acids thus is present in other than in the formor setting in which it is found in nature. However, purified hmGluRnucleic acid embraces hmGluR nucleic acid in ordinarily hmGluRexpressing cells where the nucleic acid is in a chromosomal locationdifferent from that of natural cells or is otherwise flanked by adifferent DNA sequence than that found in nature.

In particular, the invention provides a purified or isolated DNAmolecule encoding a hmGluR subtype of the invention, or a fragment ofsuch DNA. By definition, such a DNA comprises a coding single DNA, adouble stranded DNA consisting of said coding DNA and complementary DNAthereto, or this complementary (single stranded) DNA itself. Preferredis a DNA coding for the above captioned preferred hmGluR subtypes, or afragment thereof. Furthermore, the invention relates to a DNA comprisingsuch a DNA.

More specifically, preferred is a DNA coding for a hmGluR4 subtype or aportion thereof, particularly a DNA encoding the hmGluR4 subtype havingthe amino acid sequence set forth in SEQ ID NO:2, e.g. the DNA with thenucleotide sequence set forth in SEQ ID NO:1. An exemplary DNA fragmentcoding for a portion of hmGluR4 is the hmGluR4-encoding portion of cDNAcmR20 as described in the Examples.

Equally preferred is a DNA encoding a hmGluR7 subtype, particularly aDNA encoding any of the hmGluR7 subtypes having the amino acid sequencesset forth in SEQ ID NOs: 12 and 14, respectively, e.g. the DNAs with thenucleotide sequences set forth in SEQ ID NOs: 11 and 13, respectively.The invention further provides a DNA fragment encoding a portion of ahmGluR7 subtype, particularly the hmGluR7 subtypes identified aspreferred above. Exemplary hmGluR7 DNA fragments include thehmGluR7-encoding portions of cDNAs cmR2, cmR3, cmR5 and cR7PCR1, asdescribed in the Examples, or a DNA fragment which encodes substantiallythe same amino acid sequence as that encoded by the hmGluR7-encodingportion of plasmid cmR2 deposited with the DSM on Sep. 13, 1993, underaccession number DSM 8550. These DNAs encode portions of putative splicevariants of the hmGluR7 subtype described herein.

Also preferred is a DNA encoding a hmGluR6 subtype or a portion thereof,particularly a DNA encoding the portion of the hmGluR6 subtype, theamino acid sequence of which is depicted in SEQ ID NO:16, or a DNA whichencodes substantially the same amino acid sequence as that encoded bythe hmGluR6-encoding portion of plasmid cmR1 deposited with the DSM onSep. 13, 1993, under accession number DSM 8549. An exemplary DNAsequence is set forth in SEQ ID NO:15.

The nucleic acid sequences provided herein may be employed to identifyDNAs encoding further hmGluR subtypes. For example, nucleic acidsequences of the invention may be used for identifying DNAs encodingfurther hmGluR subtypes belonging to the subfamily comprising hmGluR 4.A method for identifying such DNA comprises contacting human DNA with anucleic acid probe described above and identifying DNA(s) whichhybridize to that probe.

Exemplary nucleic acids of the invention can alternatively becharacterized as those nucleic acids which encode a hmGluR subtype ofthe invention and hybridize to a DNA sequence set forth in SEQ ID NOs.1, 3, 5, 7, 9, 11, 13 or 15, or a selected portion (fragment) of saidDNA sequence. For example, selected fragments useful for hybridizationare the protein-encoding portions of said DNAs. Preferred are such DNAsencoding a hmGluR of the invention which hybridize under high-stringencyconditions to the above-mentioned DNAs.

Stringency of hybridization refers to conditions under which polynucleicacids hybrids are stable. Such conditions are evident to those ofordinary skill in the field. As known to those of skill in the art, thestability of hybrids is reflected in the melting temperature (T_(m)) ofthe hybrid which decreases approximately 1 to 1.5° C. with every 1%decrease in sequence homology. In general, the stability of a hybrid isa function of sodium ion concentration and temperature. Typically, thehybridization reaction is performed under conditions of higherstringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permithybridization of only those nucleic acid sequences that form stablehybrids in 1 M Na⁺ at 65-68° C. High stringency conditions can beprovided, for example, by hybridization in an aqueous solutioncontaining 6×SSC, 5×Denhardt's, 1% SDS (sodium dodecyl sulfate), 0.1 Na⁺pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specificcompetitor. Following hybridization, high stringency washing may be donein several steps, with a final wash (about 30 min) at the hybridizationtemperature in 0.2-0.1×SSC, 0.1% SDS.

Moderate stringency refers to conditions equivalent to hybridization inthe above described solution but at about 60-62° C. In that case thefinal wash is performed at the hybridization temperature in 1×SSC, 0.1%SDS.

Low stringency refers to conditions equivalent to hybridization in theabove described solution at about 50-52° C. In that case, the final washis performed at the hybridization temperature in 2×SSC, 0.1% SDS.

It is understood that these conditions may be adapted and duplicatedusing a variety of buffers, e.g. formamide-based buffers, andtemperatures. Denhart's solution and SSC are well known to those ofskill in the art as are other suitable hybridization buffers (see, e.g.Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning:A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, USA, or Ausubel, F. M., et al. (1993) CurrentProtocols in Molecular Biology, Greene and Wiley, USA). Optimalhybridization conditions have to be determined empirically, as thelength and the GC content of the probe also play a role.

Given the guidance of the present invention, the nucleic acids of theinvention are obtainable according to methods well known in the art. Thepresent invention further relates to a process for the preparation ofsuch nucleic acids.

For example, a DNA of the invention is obtainable by chemical synthesis,by recombinant DNA technology or by polymerase chain reaction (PCR).Preparation by recombinant DNA technology may involve screening asuitable cDNA or genomic library. A suitable method for preparing a DNAor of the invention comprises the synthesis of a number ofoligonucleotides, their amplification by PCR methods, and their splicingto give the desired DNA sequence. Suitable libraries are commerciallyavailable, e.g. the libraries employed in the Examples, or can beprepared from neural or neuronal tissue samples, e.g. hippocampus andcerebellum tissue, cell lines and the like.

For individual hmGluR subtypes (and splice variants) of the inventionthe expression pattern in neural or neuronal tissue may vary. Thus, inorder to isolate cDNA encoding a particular subtype (or splice variant),it is advantageous to screen libraries prepared from different suitabletissues or cells. As a screening probe, there may be employed a DNA orRNA comprising substantially the entire coding region of a hmGluRsubtype of the invention, or a suitable oligonucleotide probe based onsaid DNA. A suitable oligonucleotide probe (for screening involvinghybridization) is a single stranded DNA or RNA that has a sequence ofnucleotides that includes at least 14 contiguous bases that are the sameas (or complementary to) any 14 or more contiguous bases set forth inany of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 and 15. The probe may belabeled with a suitable chemical moiety for ready detection. The nucleicacid sequences selected as probes should be of sufficient length andsufficiently unambiguous so that false positive results are minimized.

Preferred regions from which to construct probes include 5′ and/or 3′coding sequences, sequences predicted to encode ligand binding sites,and the like. For example, either the full-length cDNA clones disclosedherein or fragments thereof can be used as probes. Preferably, nucleicacid probes of the invention are labeled with suitable label means forready detection upon hybridization. For example, a suitable label meansis a radiolabel. The preferred method of labelling a DNA fragment is byincorporating ³²P-labelled α-dATP with the Klenow fragment of DNApolymerase in a random priming reaction, as is well known in the art.Oligonucleotides are usually end-labeled with ³²P-labeled γ-ATP andpolynucleotide kinase. However, other methods (e.g. non-radioactive) mayalso be used to label the fragment or oligonucleotide, including e.g.enzyme labelling and biotinylation.

After screening the library, e.g. with a portion of DNA includingsubstantially the entire hmGluR-encoding sequence or a suitableoligonucleotide based on a portion of said DNA, positive clones areidentified by detecting a hybridization signal; the identified clonesare characterized by restriction enzyme mapping and/or DNA sequenceanalysis, and then examined, e.g. by comparison with the sequences setforth herein, to ascertain whether they include DNA encoding a completehmGluR (i.e., if they include translation initiation and terminationcodons). If the selected clones are incomplete, they may be used torescreen the same or a different library to obtain overlapping clones.If the library is genomic, then the overlapping clones may include exonsand introns. If the library is a cDNA library, then the overlappingclones will include an open reading frame. In both instances, completeclones may be identified by comparison with the DNAs and deduced aminoacid sequences provided herein.

Furthermore, in order to detect any abnormality of an endogenous hmGluRsubtype of the invention genetic screening may be carried out using thenucleotide sequences of the invention as hybridization probes. Also,based on the nucleic acid sequences provided herein antisense-typetherapeutic agents may be designed.

It is envisaged that the nucleic acid of the invention can be readilymodified by nucleotide substitution, nucleotide deletion, nucleotideinsertion or inversion of a nucleotide stretch, and any combinationthereof. Such modified sequences can be used to produce a mutant hmGluRsubtype which differs from the receptor subtypes found in nature.Mutagenesis may be predetermined (site-specific) or random. A mutationwhich is not a silent mutation must not place sequences out of readingframes and preferably will not create complementary regions that couldhybridize to produce secondary mRNA structures such as loops orhairpins.

The cDNA or genomic DNA encoding native or mutant hmGluR of theinvention can be incorporated into vectors for further manipulation.Furthermore, the invention concerns a recombinant DNA which is a hybridvector comprising at least one of the above mentioned DNAs.

The hybrid vectors of the invention comprise an origin of replication oran autonomously replicating sequence, one or more dominant markersequences and, optionally, expression control sequences, signalsequences and additional restriction sites.

Preferably, the hybrid vector of the invention comprises an abovedescribed nucleic acid insert operably linked to an expression controlsequence, in particular those described hereinafter.

Vectors typically perform two functions in collaboration with compatiblehost cells. One function is to facilitate the cloning of the nucleicacid that encodes the hmGluR subtype of the invention, i.e. to produceusable quantities of the nucleic acid (cloning vectors). The otherfunction is to provide for replication and expression of the geneconstructs in a suitable host, either by maintenance as anextrachromosomal element or by integration into the host chromosome(expression vectors). A cloning vector comprises the DNAs as describedabove, an origin of replication or an autonomously replicating sequence,selectable marker sequences, and optionally, signal sequences andadditional restriction sites. An expression vector additionallycomprises expression control sequences essential for the transcriptionand translation of the DNA of the invention. Thus an expression vectorrefers to a recombinant DNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into asuitable host cell, results in expression of the cloned DNA. Suitableexpression vectors are well known in the art and include those that arereplicable in eukaryotic and/or prokaryotic cells.

Most expression vectors are capable of replication in at least one classof organisms but can be transfected into another organism forexpression. For example, a vector is cloned in E. coli and then the samevector is transfected into yeast or mammalian cells even though it isnot capable of replicating independently of the host cell chromosome.DNA may also be amplified by insertion into the host genome. However,the recovery of genomic DNA encoding hmGluR is more complex than that ofexogenously replicated vector because restriction enzyme digestion isrequired to excise hmGluR DNA. DNA can be amplified by PCR and bedirectly transfected into the host cells without any replicationcomponent.

Advantageously, expression and cloning vector contain a selection genealso referred to as selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Host cells not transformed with the vectorcontaining the selection gene will not survive in the culture medium.Typical selection genes encode proteins that confer resistance toantibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate ortetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available from complex media.

Since the amplification of the vectors is conveniently done in E. coli,an E. coli genetic marker and an E. coli origin of replication areadvantageously included. These can be obtained from E. coli plasmids,such as pBR322, Bluescript vector or a pUC plasmid.

Suitable selectable markers for mammalian cells are those that enablethe identification of cells competent to take up hmGluR nucleic acid,such as dihydrofolate reductase (DHFR, methotrexate resistance),thymidine kinase, or genes confering resistance to G418 or hygromycin.The mammalian cell transfectants are placed under selection pressurewhich only those transfectants are uniquely adapted to survive whichhave taken up and are expressing the marker.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to hmGluR nucleicacid. Such promoter may be inducible or constitutive. The promoters areoperably linked to DNA encoding hmGluR by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the isolatedpromoter sequence into the vector. Both the native hmGluR promotersequence and many heterologous promoters may be used to directamplification and/or expression of hmGluR DNA. However, heterologouspromoters are preferred, because they generally allow for greatertranscription and higher yields of expressed hmGluR as compared tonative hmGluR promoter.

Promoters suitable for use with prokaryotic hosts include, for example,the β-lactamase and lactose promoter systems, alkaline phosphatase, atryptophan (trp) promoter system and hybrid promoters such as the tacpromoter. Their nucleotide sequences have been published, therebyenabling the skilled worker operably to ligate them to DNA encodinghmGluR, using linkers or adaptors to supply any required restrictionsites. Promoters for use in bacterial systems will also generallycontain a Shine-Delgarno sequence operably linked to the DNA encodinghmGluR.

HmGluR gene transcription from vectors in mammalian host cells may becontrolled by promoters compatible with the host cell systems, e.g.promoters derived from the genomes of viruses. Suitable plasmids forexpression of a hmGluR subtype of the invention in eukaryotic hostcells, particularly mammalian cells, are e.g. cytomegalovirus (CMV)promoter-containing vectors, RSV promoter-containing vectors and SV40promoter-containing vectors and MMTV LTR promoter-containing vectors.Depending on the nature of their regulation, promoters may beconstitutive or regulatable by experimental conditions.

Transcription of a DNA encoding a hmGluR subtype according to theinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector.

The various DNA segments of the vector DNA are operatively linked, i.e.they are contiguous and placed into a functional relationship to eachother.

Construction of vectors according to the invention employs conventionalligation techniques. Isolated plasmids or DNA fragments are cleaved,tailored, and religated in the form desired to generate the plasmidsrequired. If desired, analysis to confirm correct sequences in theconstructed plasmids is performed in a manner known in the art. Suitablemethods for constructing expression vectors, preparing in vitrotranscripts, introducing DNA into host cells, and performing analysesfor assessing hmGluR expression and function are known to those skilledin the art. Gene presence, amplification and/or expression may bemeasured in a sample directly, for example, by conventional Southernblotting, northern blotting to quantitate the transcription of mRNA, dotblotting (DNA or RNA analysis), in situ hybridization, using anappropriately labelled probe based on a sequence provided herein,binding assays, immunodetection and functional assays. Suitable methodsinclude those decribed in detail in the Examples. Those skilled in theart will readily envisage how these methods may be modified, if desired.

The invention further provides host cells capable of producing a hmGluRsubtype of the invention and including heterologous (foreign) DNAencoding said subtype.

The nucleic acids of the invention can be expressed in a wide variety ofhost cells, e.g. those mentioned above, that are transformed ortransfected with an appropriate expression vector. The receptor of theinvention (or a portion thereof) may also be expressed as a fusionprotein. Recombinant cells can then be cultured under conditions wherebythe protein(s) encoded by the DNA of the invention is (are) expressed.

Suitable prokaryotes include eubacteria, such as Gram-negative orGram-prositive organisms, such as E. coli, e.g. E. coli K-12 strains,DH5α and HB 101, or Bacilli. Further host cells suitable for hmGluRencoding vectors include eukaryotic microbes such as filamentous fungior yeast, e.g. Saccharomyces cerevisiae. Higher eukaryotic cells includeinsect, amphebian and vertebrate cells, particularly mammalian cells,e.g. neuroblastoma cell lines or fibroblast derived cell lines. Examplesof preferred mammalian cell lines are e.g. HEK 293 cells, CHO cells, CV1cells, BHK cells, L cells, LLCPK-1 cells, GH3 cells, L cells and COScells. In recent years propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. The host cells referredto in this application comprise cells in in vitro culture as well ascells that are within a host animal.

Suitable host cells for expression of an active recombinant hmGluR ofthe invention advantageously express endogenous or recombinantG-proteins. Preferred are cells producing little, if any, endogenousmetabotropic glutamate receptor. DNA may be stably incorporated into thecells or may be transiently expressed according to conventional methods.

Stably transfected mammalian cells may be prepared by transfecting cellswith an expression vector having a selectable marker gene, and growingthe transfected cells under conditions selective for cells expressingthe marker gene. To prepare transient transfectants, mammalian cells aretransfected with a reporter gene to monitor transfection efficiency.

To produce such stably or transiently transfected cells, the cellsshould be transfected with a sufficient amount of hmGluR-encodingnucleic acid to form hmGluR of the invention. The precise amounts of DNAencoding hmGluR of the invention may be empirically determined andoptimized for a particular cell and assay.

A DNA of the invention may also be expressed in non-human transgenicanimals, particularly transgenic warm-blooded animals. Methods forproducing transgenic animals, including mice, rats, rabbits, sheep andpigs, are known in the art and are disclosed, for example by Hammer etal. (Nature 315, 680-683, 1985). An expression unit including a DNA ofthe invention coding for a hmGluR together with appropriately positionedexpression control sequences, is introduced into pronuclei of fertilizedeggs. Introduction may be achieved, e.g. by microinjection. Integrationof the injected DNA is detected, e.g. by blot analysis of DNA fromsuitable tissue samples. It is preferred that the introduced DNA beincorporated into the germ line of the animal so that it is passed tothe animal's progeny. Preferably, a transgenic animal is developped bytargeting a mutation to disrupt a hmGluR sequence. Such an animal isuseful e.g. for studying the role of a metabotropic receptor inmetabolism.

Furthermore, a knock-out animal may be developed by introducing amutation in the hmGluR sequence, thereby generating an animal which doesnot express the functional hmGluR gene anymore. Such knock-out animal isuseful e.g. for studying the role of metabotropic receptor inmetabolism. methods for producing knock-out mice are known in the art.

Host cells are transfected or transformed with the above-captionedexpression or cloning vectors of this invention and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. Heterologous DNA may be introduced into host cells byany method known in the art, such as transfection with a vector encodinga heterologous DNA by the calcium phosphate coprecipitation technique,by electroporation or by lipofectin-mediated. Numerous methods oftransfection are known to the skilled worker in the field. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs in the host cell. Transformation isachieved using standard techniques appropriate to the particular hostcells used.

Incorporation of cloned DNA into a suitable expression vector,transfection of eukaryotic cells with a plasmid vector or a combinationof plasmid vectors, each encoding one or more distinct genes or withlinear DNA, and selection of transfected cells are well known in the art(see, e.g. Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press).

Transfected or transformed cells are cultured using media and culturingmethods known in the art, preferably under conditions, whereby hmGluRencoded by the DNA is expressed. The composition of suitable media isknown to those in the art, so that they can be readily prepared.Suitable culturing media are also commercially available.

While the DNA provided herein may be expressed in any suitable hostcell, e.g. those referred to above, preferred for expression of DNAencoding functional hmGluR are eukaryotic expression systems,particularly mammalian expression systems, including commerciallyavailable systems and other systems known to those of skill in the art.

Human mGluR DNA of the invention is ligated into a vector, andintroduced into suitable host cells to produce transformed cell linesthat express a particular hmGluR subtype of the invention, or specificcombinations of subtypes. The resulting cell line can then be producedin amounts sufficient for reproducible qualitative and quantitativeanalysis of the effects of a receptor agonist, antagonist or allostericmodulator. Additionally, mRNA may be produced by in vitro transcriptionof a DNA encoding a subtype of the invention. This mRNA may be injectedinto Xenopus oocytes where the mRNA directs the synthesis of the activereceptor subtype. Alternatively, the subtype-encoding DNA can bedirectly injected into oocytes. The transfected mammalian cells orinjected oocytes may then be employed in an drug screening assayprovided hereinafter. Such drugs are useful in diseases associated withpathogenesis of a hmGluR subtype of the invention. Such diseases includediseases resulting from excessive action of glutamate preferentiallymediated by hmGluRs, such as stroke, epilepsy and chronicneurogenerative diseases. Particularly useful for assessing the specificinteraction of compounds with specific hmGluR subtypes are stablytransfected cell lines expressing a hmGluR of the invention.

Thus host cells expressing hmGluR of the invention are useful for drugscreening and it is a further object of the present invention to providea method for identifying a compound or signal which modulates theactivity of hmGluR, said method comprising exposing cells containingheterologous DNA encoding hmGluR of the invention, wherein said cellsproduce functional hmGluR, to at least one compound or signal whoseability to modulate the activity of said hmGluR is sought to bedetermined, and thereafter monitoring said cells for changes caused bysaid modulation. Such an assay enables the identification of agonists,antagonists and allosteric modulators of a hmGluR of the invention.

In a further aspect, the invention relates to an assay for identifyingcompounds which modulate the activity of a hmGluR subtype of theinvention, said assay comprising:

contacting cells expressing an active hmGluR subtype of the inventionand containing heterologous DNA encoding said hmGluR subtype with atleast one compound to be tested for its ability to modulate the activityof said receptor, and

analysing cells for a difference in second messenger level or receptoractivity.

In particular, the invention covers an assay for identifying compoundswhich modulate the activity of a hmGluR subtype of the invention, saidassay comprising:

contacting cells expressing active hmGluR of the invention andcontaining heterologous DNA encoding said hmGluR subtype with at leastone compound to be tested for its ability to modulate the activity ofsaid receptor, and

monitoring said cells for a resulting change in second messengeractivity.

The result obtained in the assay is compared to an assay suitable as anegative control.

Assay methods generally require comparison to various controls. A changein receptor activity or in second messenger level is said to be inducedby a test compound if such an effect does not occur in the absence ofthe test compound. An effect of a test compound on a receptor subtype ofthe invention is said to be mediated by said receptor if this effect isnot observed in cells not expressing the receptor.

As used herein, a compound or signal that modulates the activity of ahmGluR of the invention refers to a compound or signal that alters theresponse pathway mediated by said hmGluR within a cell (as compared tothe absence of said hmGluR). A response pathway is activated by anextracellular stimulus, resulting in a change in second messengerconcentration or enzyme activity, or resulting in a change of theactivity of a membrane-bound protein, such as a receptor or ion channel.A variety of response pathways may be utilized, including for example,the adenylate cyclase response pathway, the phospholipaseC/intracellular calcium ion response pathway or coupling to an ionchannel. Assays to determine adenylate cyclase activity are well knownin the art, and include e.g. the assay disclosed by Nakajima et al., J.Biol. Chem. 267, 2437-2442 (1992)).

Thus cells expressing hmGluR of the invention may be employed for theidentification of compounds, particularly low molecular weight moleculescapable of acting as glutamate agonists or antagonists. Preferred arelow molecular weight molecules of less than 1,000 Dalton. Within thecontext of the present invention, an agonist is understood to refer to amolecule that is capable of interacting with a receptor, thus mimickingthe action of L-glutamate. In particular, a glutamate agonist ischaracterized by its ability to interact with a hmGluR of the invention,and thereby increasing or decreasing the stimulation of a responsepathway within a cell. For example, an agonist increases or decreases ameasurable parameter within the host cell, such as the concentration ofa second messenger, as does the natural ligand increase or decrease saidparameter. For example, in a suitable test system, wherein hmGluR of theinvention is negatively coupled to adenylate cyclase, e.g. CHO or BHKcells expressing a hmGluR of the invention, such an agonist is capableof modulating the function of said hmGluR such that the intracellularconcentration of cAMP is decreased.

By contrast, in situations where it is desirable to tone down theactivity of hmGluR, antagonizing molecules are useful. Within thecontext of the present invention, an antagonist is understood to referto a molecule that is capable of interacting with a receptor or withL-glutamate, but which does not stimulate a response pathway within acell. In particular, glutamate antagonists are generally identified bytheir ability to interact with a hmGluR of the invention, and therebyreduce the ability of the natural ligand to stimule a response pathwaywithin a cell, e.g. by interfering with the binding of L-glutamate to ahmGluR of the invention or by inhibiting other cellular functionsrequired for the activity of hmGluR. For example, in a suitable assay,e.g. an assay involving CHO or BHK cells expressing a hmGluR subtype ofthe invention, a glutamate antagonist is capable of modulating theactivity of a hmGluR of the invention such that the ability of thenatural ligand to decrease the intracellular cAMP concentration isweakened. Yet another alternative to achieve an antagonistic effect isto rely on overexpression of antisense hmGluR RNA. Preferred is anagonist or antagonist selectively acting on a receptor of the hmGluR4subfamily, e.g. hmGluR4, hmGluR6 or hmGluR7. Particularly useful is anagonist or antagonist specifically modulating the activity of aparticular hmGluR subtype without affecting the activity of any othersubtype.

An allosteric modulator of a hmGluR of the invention interacts with thereceptor protein at another site than L-glutamate, thus acting asagonist or antagonist. Therefore, the screening assays decribed hereinare also useful for detecting an allosteric modulator of a receptor ofthe invention. For example, an allosteric modulator acting as agonistmay enhance the specific interaction between a hmGluR of the inventionand L-glutamate. If an allosteric modulator acts as an antagonist, itmay e.g. interact with the receptor protein in such a way that bindingof the agonist is functionally less effective.

An in vitro assay for a glutamate agonist or antagonist may require thata hmGluR of the invention is produced in sufficient amounts in afunctional form using recombinant DNA methods. An assay is then designedto measure a functional property of the hmGluR protein, e.g. interactionwith a glutamatergic ligand. Production of a hmGluR of the invention isregarded as occurring in sufficient amounts, if activity of saidreceptor results in a measurable response.

For example, mammalian cells, e.g. HEK293 cells, L cells, CHO-K1 cells,LLCPK-1 cells or GH3 cells (available e.g. from the American Tissue TypeCulture Collection) are adapted to grow in a glutamate reduced,preferably a glutamate free, medium. A hmGluR expression plasmid, e.g. aplasmid described in the Examples, is transiently transfected into thecells, e.g. by calcium-phosphate precipitation (Ausubel, F. M., et al.(1993) Current Protocols in Molecular Biology, Greene and Wiley, USA).Cell lines stably expressing a hmGluR of the invention may be generatede.g. by lipofectin-mediated transfection with hmGluR expression plasmidsand a plasmid comprising a selectable marker gene, e.g. pSV2-Neo(Southern and Berg, J. Mol. Appl. Genet. 1, 327-341 (1982)), a plasmidvector encoding the G-418 resistence gene. Cells surviving the selectionare isolated and grown in the selection medium. Resistant clonal celllines are analyzed, e.g. for immunoreactivity with subtype-specifichmGluR antibodies or by assays for hmGluR functional responses followingagonist addition. Cells producing the desired hmGluR subtype are used ina method for detecting compounds binding to a hmGluR of the invention orin a method for identifying a glutamate agonist or antagonist.

In a further embodiment, the invention provides a method for identifyingcompounds binding to a hmGluR subtype, said method comprising employinga hmGluR subtype of the invention in a competitive binding assay. Theprinciple underlying a competitive binding assay is generally kown inthe art. Briefly, binding assays according to the invention areperformed by allowing the compound to be tested for its hmGluR bindingcapability to compete with a known, suitably labeled, glutamatergicligand for the binding site at the hmGluR target molecule. A suitablylabeled ligand is e.g. a radioactively labeled ligand, such as[³H]glutamate, or a ligand which can be detected by its opticalproperties, such as absorbance or fluorescence. After removing unboundligand and test compound the amount of labeled ligand bound to hmGluR ismeasured. If the amount of labeled ligand is reduced in the presence ofthe test compound this compound is said to be bound to the targetmolecule. A competitive binding assay may be performed e.g. withtransformed or transfected host cells expressing a hmGluR of theinvention or a membraneous cellular fraction comprising a hmGluR of theinvention.

Compound bound to the target hmGluR may modulate the functionalproperties of hmGluR and may thereby be identified as a glutamateagonist or antagonist in a functional assay.

Functional assays are used to detect a change in the functional activityof a hmGluR of the invention, i.e. to detect a functional response, e.g.as a result of the interaction of the compound to be tested with saidhmGluR. A functional response is e.g. a change (difference) in theconcentration of a relevant second messenger, or a change in theactivity of another membrane-bound protein influenced by the receptor ofthe invention within cells expressing a functional hmGluR of theinvention (as compared to a negative control). Those of skill in the artcan readily identify an assay suitable for detecting a change in thelevel of an intracellular second messenger indicative of the expressionof an active hmGluR (functional assay). Examples include cAMP assays(see, e.g. Nakajima et al., J. Biol. Chem. 267, 2437-2442 (1992), cGMPassays (see, e.g. Steiner et al., J. Biol. Chem. 247, 1106-1113 (1972)),phosphatidyl inositol (PI) turnover assays (Nakajima et al., J. Biol.Chem. 267, 2437-2442 (1992)), calcium ion flux assays (Ito et al., J.Neurochem. 56, 531-540 (1991)), arachidonic acid release assays (see,e.g. Felder et al., J. Biol. Chem. 264, 20356-20362 (1989)), and thelike.

More specifically, according to the invention a method for detecting aglutamate agonist comprises the steps of (a) exposing a compound to ahmGluR subtype of the invention coupled to a response pathway, underconditions and for a time sufficient to allow interaction of thecompound with the receptor and an associated response through thepathway, and (b) detecting an increase or decrease in the stimulation ofthe response pathway resulting from the interaction of the compound withthe hmGluR subtype, relative to the absence of the tested compound andtherefrom determining the presence of a glutamate agonist.

A method for identifying a glutamate antagonist comprises the steps of(a) exposing a compound in the presence of a known glutamate agonist toa hmGluR subtype of the invention coupled to a response pathway, underconditions and for a time sufficient to allow interaction of the agonistwith the receptor and an associated response through the pathway, and(b) detecting an inhibition of the stimulation of the response pathwayby the agonist resulting from the interaction of the compound with thehmGluR subtype, relative to the stimulation of the response pathway bythe glutamate agonist alone and therefrom determining the presence of aglutamate antagonist. Inhibition may be detected, e.g. if the testcompound competes with the glutamate agonist for the hmGluR of theinvention. Compounds which may be screened utilizing such method aree.g. blocking antibodies specifically binding to the hmGluR subtype.Furthermore, such an assay is useful for the screening for compoundsinteracting with L-glutamate, e.g. soluble hmGluR fragment s comp risingpart or all of the ligand binding domain.

Preferentially, interaction of an agonist or antagonist with a hmGluR ofthe invention denotes binding of the agonist or antagonist to saidhmGluR.

As employed herein, conditions and times sufficient for interaction of aglutamate agonist or antagonist candidate to the receptor will vary withthe source of the receptor, however, conditions generally suitable forbinding occur between about 4° C. and about 40° C., preferably betweenabout 4° C. and about 37° C., in a buffer solution between 0 and 2 MNaCl, preferably between 0 and 0.9 M NaCl, with 0.1 M NaCl beingparticularly preferred, and within a pH range of between 5 and 9,preferably between 6.5 and 8. Sufficient time for the binding andresponse will generally be between about 1 ms and about 24 h afterexposure.

Within one embodiment of the present invention, the response pathway isa membrane-bound adenylate cyclase pathway, and, for an agonist, thestep of detecting comprises measuring a reduction or increase,preferably a reduction, in cAMP production by the membrane-boundadenylate cyclase response pathway, relative to t he AMP production inthe relevant control setup. For the purpose of the present invention, itis preferred that the reduction or increase in cAMP production beequivalent or greater than the reduction or increase induced byL-glutamate applied at a concentration corresponding to its IC₅₀concentration. For an antagonist, the step of detecting comprisesmeasuring in the presence of the antagonist a smaller L-glutamateinduced decrease or increase in cAMP production by the membrane-boundadenylate cyclase response pathway, as compared to the cAMP productionin the absence of the antagonist. The measurement of cAMP may beperformed after cell destruction or by a cAMP sensitive molecular probeloaded into the cell, such as a fluorescent dye, which changes itsproperties, e.g. its fluorescent properties, upon binding of cAMP.

Cyclic AMP production may be measured using methods well known in theart, including for instance, methods described by Nakajima et al.,supra, or using commercially available kits, e.g. kits comprisingradiolabeled cAMP, e.g. [¹²⁵I]cAMP or [³H]cAMP. Exemplary kits are theScintillation Proximity Assay Kit by Amersham, which measures theproduction of cAMP by competition of iodinated-cAMP with cAMPantibodies, or the Cyclic AMP [³H] Assay Kit by Amersham.

In assay systems using cells expressing receptor subtypes that arenegatively coupled to the adenylate cyclase pathway, i.e. which cause adecrease in cAMP upon stimulation and an increase in cAMP upon reductionof stimulation, it is preferred to expose the cells to a compound whichreversibly or irreversibly stimulates the adenylate cyclase, e.g.forskolin, or which is a phosphodiesterase inhibitor, such asisobutylmethylxanthine (IBMX), prior to addition of the (potential)receptor agonist or antagonist.

Within another embodiment of the invention, the response pathway is thePI hydrolysis/Ca²⁺ mobilization pathway. Such an assay for determiningthe specific interaction of a test compound with a hmGluR subtype of theinvention may be functionally linked to changes in the intracellularcalcium ion (Ca²⁺) concentration. Several methods for determining achange in the intracellular concentration of Ca²⁺ are known in the art,e.g. a method involving a calcium ion sensitive fluoroscent dye, such asfura-2 (see Grynkiewisz et al., J. Biol. Chem. 260, 3440-3450, 1985),fluo-3 or Indo-1, such as the calcium fluor QuinZ method describe byCharest et al. (J. Biol. Chem. 259, 8679-8773 (1993)), or the aequorinphotoprotein method described by Nakajima-Shimada (Proc. Natl Acad. Sci.USA 88, 6878-6882 (1991)). In one embodiment of the invention,intracellular calcium ion concentration is measured by microfluoremetryin recombinant cells loaded with calcium sensitive fluorescent dyesfluo-3 or fura-2. These measurements may be performed using cells grownin a coverslip allowing the use of an inverted microscope andvideo-imaging technologies or a fluorescence photometer to measurecalcium concentrations at the single cell level. For both approaches,cells transformed with a hmGluR expressing plasmid have to be loadedwith the calcium indicator. To this end, the growth medium is removedfrom the cells and replaced with a solution containing fura-2 or fluo-3.The cells are used for calcium measurements preferentially during thefollowing 8h. The microfluorometry follows standard procedures.

Ca²⁺ signals resulting from functional interaction of compounds with thetarget molecule can be transient if the compound is applied for alimited time period, e.g. via a perfusion system. Using transientapplication several measurements can be made with the same cellsallowing for internal controls and high numbers of compounds tested.

Functional coupling of a hmGluR of the invention to Ca²⁺ signaling maybe achieved, e.g. in CHO cells, by various methods:

(i) coexpression of a recombinant hmGluR of the invention and arecombinant voltage-gated cation channel, activity of which isfunctionally linked to the activity of the hmGluR;

(ii) expression of a chimeric hmGluR receptor, which directly stimulatesthe PI/Ca²⁺ pathway;

(iii) coexpression of a recombinant hmGluR of the invention with arecombinant Ca²⁺-permeable cAMP dependent cation channel.

In other expression systems functional coupling of a hmGluR to Ca²⁺signalling may be achieved by transfection of a hmGluR of the inventionif these cells naturally express (i) voltage gated Ca channels, activityof which is functionally linked to activity of mGluRs or (ii)Ca²⁺-permeable cAMP dependent ion channels. For example, GH3 cells whichnaturally express voltage-gated Ca channels, directly allow applicationof Ca²⁺ assays to test for hmGluR functional activity by cotransfectionof hmGluRs.

Further cell-based screening assays can be designed e.g. by constructingcell lines in which the expression of a reporter protein, i.e. an easilyassayable protein, such as β-galactosidase, chloramphenicolacetyltransferase (CAT) or luciferase, is dependent on the function of ahmGluR of the invention. For example, a DNA construct comprising a cAMPresponse element is operably linked to a DNA encoding luciferase. Theresulting DNA construct comprising the enzyme DNA is stably transfectedinto a host cell. The host cell is then transfected with a second DNAconstruct containing a first DNA segment encoding a hmGluR of theinvention operably linked to additional DNA segments necessary for theexpression of the receptor. For example, if binding of a test compoundto the hmGluR of the invention results in elevated cAMP levels, theexpression of luciferase is induced or decreased, depending on thepromoter chosen. The luciferase is exposed to luciferin, and the photonsemitted during oxidation of luciferin by the luciferase is measured.

The drug screening assays provided herein will enable identification anddesign of receptor subtype-specific compounds, particularly ligandsbinding to the receptor protein, eventually leading to the developmentof a disease-specific drug. If designed for a very specific interactionwith only one particular hmGluR subtype (or a predetermined selection ofhmGluR subtypes) such a drug is most likely to exhibit fewer unwantedside effects than a drug identified by screening with cells that expressa(n) (unknown) variety of receptor subtypes. Also, testing of a singlereceptor subtype of the invention or specific combinations of differentreceptor subtypes with a variety of potential agonists or antagonistsprovides additional information with respect to the function andactivity of the individual subtypes and should lead to theidentification and design of compounds that are capable of very specificinteraction with one or more receptor subtypes.

In another embodiment the invention provides polyclonal and monoclonalantibodies generated against a hmGluR subtype of the invention. Suchantibodies may useful e.g. for immunoassays includingimmunohistochemistry as well as diagnostic and therapeutic applications.For example, antibodies specific for the extracellular domain, orportions thereof, of a particular hmGluR subtype can be applied forblocking the endogenous hmGluR subtype.

The antibodies of the invention can be prepared according to methodswell known in the art using as antigen a hmGluR subtype of theinvention, a fragment thereof or a cell expressing said subtype orfragment. The antigen may represent the active or inactive form of thereceptor of the invention. Antibodies may be capable of distinguishingbetween the active or inactive form. Factors to consider in selectingsubtype fragments as antigens (either as synthetic peptide or as fusionprotein) include antigenicity, accessibility (i.e. extracellular andcytoplasmic domains) and uniqueness to the particular subtype.

Particularly useful are antibodies selectively recognizing and bindingto receptor subtypes of the above described subfamily without binding toa subtype of another subfamily and antibodies selectively recognizingand binding to one particular subtype without binding to any othersubtype.

The antibodies of the invention can be administered to a subject in needthereof employing standard methods. One of skill in the art can readilydetermine dose forms, treatment regimens etc, depending on the mode ofadministration employed.

The invention particularly relates to the specific embodiments asdescribed in the Examples which serve to illustrate the presentinvention but should not be construed as a limitation thereof.

Abbreviations: hmGluR=human metabotropic glutamate receptor,nt=nucleotide

EXAMPLE 1

cDNA Encoding hmGluR4

Human mGluR4 cDNA clones are isolated from human fetal brain and humancerebellum cDNA libraries by low stringency hybridization using aradiolabeled rat mGluR4 probe generated by PCR from rat brain cDNA.

1.1 Preparation of Poly(A)⁺ RNA from Rat Forebrain

Adult male Sprague-Dawley rats are killed by suffocation, theirforebrain is removed and immediately frozen in liquid N₂. Total RNA isisolated using the guanidinium thio-cyanate-procedure (Chomczynski andSacchi (1987), Anal. Biochem. 162, 156-159). Enrichment of poly(A)⁺ RNAis achieved by affinity chromatography on oligo(dT)-cellulose accordingto standard procedures (Sambrook, J., Fritsch, E. F. and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, USA).

1.2 First Strand cDNA Synthesis for PCR

Poly(A)⁺RNA (mRNA) is reverse-transcribed into DNA by Moloney MurineLeukemia Virus Reverse Transcriptase (M-MLV RT, BRL). 50 μl reactionsare set up as follows: 10 μg of rat forebrain poly(A)⁺RNA in 10 μlsterile H₂O are heated to 70° C. for 10 min and then quickly chilled onice. Then, 10 μl 5×reaction buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl,15 mM MgCl₂), 5 μl 0.1M dithiothreitol, 5 μl mixed dNTP (10 mM each ofdATP, dCTP, dGTP, dTTP, Pharmacia), 1.25 μl oligo-dT₁₂₋₁₈ (2 mg/ml,Pharmacia), 2.5 μl RNAsin (40 U/μl, Promega), 12.25 μl sterile H₂O and 4μl (200 U/μl) M-MLV RT are added. The reaction is carried out at 37° C.for 60 min.

1.3 PCR Conditions for Generating the Rat mGluR4 Fragment

The oligodeoxynucleotide primers used for PCR are synthesized by thephosphoramidite method. Sequences are listed in Table 1.

TABLE 1 P1: 5′-GTCAAGGCCTCGGGCCGGGA- 3′ (SEQ ID NO:17) corresponding tobp 1921-1940 of rat mGluR4 cDNA (Tanabe, et al., (1992), Neuron 8,169-179) P2: 5′-CTAGATGGCATGGTTGGTGTA-3′ (SEQ ID NO:18) corresponding tobp 2788-2808 of rat mGluR4 cDNA (Tanabe, et al., (1992), Neuron 8,169-179)

Standard PCR-conditions for a 100 μl reaction mixture are: 30 ng of ratforebrain cDNA, 50 pmol each of primers P1 and P2, 200 μmol each of thefour deoxynucleoside triphosphates dATP, dCTP, dGTP and dTTP, 10% DMSOin PCR-buffer (10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, 10 mMβ-mercaptoethanol, 0.05% Tween (w/v), 0.05% NP-40 (w/v)), and 0.5 UAmpliTaq Polymerase (Perkin Elmer Cetus). The amplification is performedusing the following conditions: 30 sec denaturing at 93° C., 1 min 30sec annealing at 56° C., and 3 min extension at 72° C., for a total of40 cycles. Initial denaturation is carried out for 4 min at 94° C.

1.4 Subcloning of the Rat mGluR4 PCR Fragment

Restriction endonuclease digestions, use of modifying enzymes, vectorpreparation (dephosphorylation, gel purification), ligations,transformation of E. coli, and plasmid DNA preparations are performedaccording to standard procedures (Sambrook, et al. (1989), supra).

The PCR fragment (888 bp) obtained according to the procedure describedin 1.3 is ligated into the SmaI site of the Bluescript SK⁺ plasmid(Stratagene, La Jolla, USA). The fragment inserted into the Bluescriptvector is sequenced from both ends using T7 and T3 primers (Stratagene,La Jolla, USA).

1.5 Preparation of a Radiolabeled Probe

20-50 ng of the PCR generated rat mGluR4 fragment are gel purified and³²P-labeled by random priming using a DNA Labeling Kit (BoehringerMannheim).

1.6 cDNA Library Screening

About 1×10⁶ phages from a human fetal brain library (λZAPII, Stratagene,La Jolla, USA), human hippocampus (ZAP, Stratagene, La Jolla, USA), anda human cerebellum cDNA library (λZAP, Stratagene) are screened forhybridization to the rat mGluR4 fragment. Hybridization is performed in5×SSC, 0.02% (w/v) Ficoll (Type 400), 0.02% (w/v) Polyvinylpyrrolidone,0.1% (w/v) SDS, 50 μg/ml Herring Testis DNA. Prehybridization is carriedout between 30 min to 3 hours at 58° C. Hybridization is carried out atlow stringency at 58° C. overnight in the same solution containing the³²P-labeled fragment at a concentration of 1-3×10⁵ cpm/ml. Washes aredone three times for 20 min each at 58° C. in 2×SSC/0.1% SDS.

Phages hybridizing to the rat mGluR4 probe are purified by a second andthird round of screening under the conditions described above. The cDNAinserts harbored by the purified phages are rescued by in vivo excisionusing the ExAssist/SOLR system (Stratagene, La Jolla, USA).

1.7 Characterization of Isolated cDNA Clones

Several cDNA inserts are characterized by restriction enzyme mapping andDNA sequence analysis. One of these clones, cDNA cmR20 (isolated fromhuman cerebellar library) contains an insert of approximately 3.3 kb.Sequence analysis of cmR20 indicates that it contains almost thecomplete coding region of human mGluR4 including a translationtermination codon (nt 158 to 2739, cf. SEQ ID NO:1) as well asapproximately 750 nt of 3′ untranslated region. The 5′ end including thetranslational start codon is lacking.

1.8 Isolation of the 5′ End of Human mGluR4

To complete the coding region of human mGluR4 PCR reactions are carriedout using human genomic DNA or first strand cDNA of human brain RNA as atemplate. The sense primer P3 corresponds to the 5′ end of the ratmGluR4 cDNA, the antisense primer P4 to nt 440-459 of the rat mGluR4cDNA.

TABLE 2 P3: 5′-GCGCTGCAGGCGGCCGC AGGGCCTGCTAGGGCTAGGAGCGGGGC-3′ (SEQ IDNO:19) corresponding to nt 11-37 of rat mGluR4 CDNA (Tanabe, et al.,(1992), Neuron 8, 169-179) P4: 5′-GCGGAATTC CCTCCGTGCCGTCCTTCTCG-3′ (SEQID NO:20) corresponding to nt 440-459 of rat mGluR4 cDNA (Tanabe, etal., (1992), Neuron 8, 169-179) Additional sequences are underlined,sites for restriction enzymes are indicated in boldface.

PCR reactions for a 100 μl reaction mixture are: 400 ng of human genomicDNA, 1 μM of each primer, 2 mM of each deoxynucleoside triphosphate(dATP, dCTP, dGTP and dTTP) in PCR-buffer (10 mM Tris-HCl, pH 8.3, 1.5mM MgCl₂, 50 mM KCl, and 2 U AmpliTaq Polymerase. The amplification isperformed using, the following conditions: 1 min denaturation at 95° C.,1 min annealing at 56° C., and 1 min extension at 72° C., for a total of32 cycles. Initial denaturation is carried out for 3 min at 94° C.

Products of several independent PCRs are digested with restrictionenzymes PstI and EcoRI, gel purified, and ligated into the PstI/EcoRIsites of pBluescript SK (Stratagene). Subcloned fragments of severalindependent PCRs are analyzed by DNA sequence analysis (cR4PCR1-4).Sequence analysis reveals that clone cR4PCR2 encodes 380 nt of hmGluR4coding region including the translation initiation codon (nt 1-380, cf.SEQ ID NO:1). cR4PCR2 overlaps at the 3′ end for 223 nt with cmR20.

The complete deduced amino acid sequence of the hmGluR4 protein is setforth in SEQ ID NO:2.

EXAMPLE 2

cDNA Clones Encoding hmGluR7

Screening of human fetal brain and human cerebellum cDNA libraries bylow-stringency hybridization using radiolabeled rat mGluR4 fragment (asdescribed in 1.5 and 1.6) allows the isolation of cDNA clones thatidentify the human metabotropic glutamate receptor subtype mGluR7.Characterization of cDNA clones by DNA sequence analysis reveals thatisolated cDNAs represent at least two apparent splice variants of humanmGluR7 mRNA.

cDNA cmR2 (isolated from human fetal brain cDNA library) has a size of3804 nt. Clone cmR2 contains 2604 nt of hmGluR7 coding sequenceincluding a translation termination codon followed by 1200 nt of 3′untranslated sequence (cf. SEQ ID NO:3).

cDNA cmR3 (isolated from human hippocampus cDNA library) has a size of1399 nt (SEQ ID NO:5). cmR3 contains 270 nt of the hmGluR7 3′ end codingregion including a translation termination stop codon (the deduced aminoacid sequence is set forth in SEQ ID NO:6) followed by 1129 nt of 3′untranslated sequence. The sequence of cmR3 is completely contained incmR2 but differs from cmR2 by deletion of the 92 nucleotides extendingfrom the nt at position 2534 to the nt at position 2625 in SEQ ID NO:3).This apparent splice variant of hmGluR7 generates a different 3′ end ofthe deduced hmGluR7 amino acid sequence.

cDNA cmR5 (isolated from human fetal brain cDNA library) has a size of1588 nt (SEQ ID NO:7). cDNA cmR5 overlaps 1424 nt with cDNA cmR2. Itdiverges at the 3′ end exactly at the position of the92-nt-insertion/deletion of cmR2/cmR3. Additional 164 nt of cmR5 eitherencode intronic sequences as indicated by presence of a conserved splicedonor sequence immediately following the site of cmR5 and cmR2/cmR3sequence divergence, or represent a third splice variant.

The 5′ end coding region of hmGluR7 DNA missing in cDNA clones cmR2,cmR3, and cmR5, is isolated by a combination of genomic libraryscreening and PCR techniques. A Lamda-Fix genomic library (Stratagene)is screened with a EcoRI/SmaI restriction fragment comprising nt 1-1304of cDNA cmR2 under high stringency hybridization conditions as describedin Sambrook, et al. (1989), supra. Lambda clones hybridizing to the 5′end of cDNA clone cmR2 are purified and analyzed by restriction analysesand DNA sequencing. The complete 5′ end of the coding region of humanmGluR7 including the ATG translation initiation codon is amplified byPCR from human brain cDNA using primer sequences derived from clonedgenomic fragments. The PCR fragments has a size of 557 nt. It isdesignated as cR7PCR1 and depicted as SEQ ID NO:9. The deduced aminoacid sequence is set forth in SEQ ID NO:10. cR7PCR1 overlaps at the 3′end with cmR2 for 392 nt.

The DNA sequences coding for the complete hmGluR7a and b proteins areset forth in SEQ ID NOs:11 and 13, respectively. The deduced amino acidsequences are given in SEQ ID NOs:12 and 14, respectively. Comparison ofthe deduced amino acid sequences reveals approximately 70% sequenceidentity to the hmGluR4 subtype of Example 1.

EXAMPLE 3

cDNA Encoding Partial hmGluR6

A single cDNA clone, cmR1, with an insert of 1.0 kb is isolated from ahuman hippocampus library by low stringency hybridization using thehmGluR fragment as described above in example 1.5 and 1.6. Approximately630 nucleotides are homologous to human mGluR4. Additional sequences atthe 5′ and 3′ end of cmR1 apparently encode intronic sequences asindicated by the presence of putative splice donor and splice acceptorsite sequences. cDNA cmR1 identifies a portion of the human metabotropicglutamate receptor subtype hmGluR6 (SEQ ID NOs. 15). The deduced aminoacid sequence is set forth in SEQ ID NO:16.

The complete coding region of hmGluR6 is isolated by screening of cDNAand genomic libraries under high stringency conditions with cDNA cmR1 asa probe. Comparison of the deduced amino acid sequences revealsapproximately 70% sequence identity to hmGluR4 of Example 1.

EXAMPLE 4

Expression of hmGluR cDNAs in Mammalian Cells

4.1 Receptor Expression Plasmids

cDNAs encoding the above full-length hmGluR4, hmGluR6, and hmGluR7proteins are generated from cDNA fragments and ligated into mammalianexpression vectors based on constitutive promoters (CMV, SV40, RSV) orinducible promoters. Examples are pBK-CMV (Stratagene), pBK-RSV(Stratagene), pCMV-T7 (Sibia, Inc.) and pICP4 (Novagen, USA).

The full-length cDNA encoding the hmGluR4 subtype is incorporated intothe mammalian expression vector pBK-CMV by ligating the hmGluR4 5′ endfragment (clone cR4PCR2) with cDNA cmR20 at the unique XhoI site that islocated at nt 346-351 of the hmGluR4 cDNA. Specifically, plasmidpBK-CMV-hmGluR4 is generated by three-way-ligation of the NotI/XhoIfragment of cR4PCR2, the XhoI/NotI fragment of cDNA cmR20 and the NotIdigested vector pBK-CMV. Plasmid pCMV-T7-hmGluR4 is generated bythree-way-ligation of the PstI/XhoI fragment of cR4PCR4, the XhoI/EcoRIfragment of cmR20 and the PstI/EcoRI digested vector pCMV-T7-2. Bothexpression constructs contain the complete coding region of the hmGluR4as well as approximately 750 nt of 3′ untranslated sequences.

Full-length cDNAs representing the two hmGluR7 splice variants,designated hmGluR7a (SEQ ID NO:12) and hmGluR7b SEQ ID NO:14), areincorporated in pCMV-T7-2 (SIBIA Inc.) using the overlapping cDNA clonescmR2, cmR3 and hcR7PCR1. A full-length hmGluR7b expression construct,designated pCMV-T7-hmGluR7b, is prepared by three-way-ligation of thePstV/BsaI fragment of hcR7PCR 1, the BsaV/EagI fragment of cmR2 and thePstI/NotI of pCMV-T7-2. Plasmid pCMV-T7-hmGluR7b contains the completecoding region of hmGluR7b and 191 nt of 3′ untranslated sequences. Toconstruct a full-length hmGluR7a expression construct, designatedpCMV-T7-hmGluR7a, a 370 bp HindIII/EagI fragment of cmR2 is exchangedwith the corresponding fragment of cmR3. The BsaI/EagI fragment of theresulting clone is used for a three-way-ligation as describe above.

Plasmid pBK-CMV-hmGluR6 is generated analogously using conventionaltechniques (Sambrook et al. supra).

4.2 Transfection of Mammalian Cells

Mammalian cells (e.g. CHO-K1, GH3; American Tissue Type CultureCollection) are adapted to grow in glutamate free medium (Dulbecco'smodified Eagle's medium lacking L-glutamate and containing a reducedconcentration of 2 mM L-glutamine, supplemented with 0.046 mg/ml prolineand 10% dialyzed fetal bovine serum, Gibco-BRL). HmGluR expressionplasmids are transiently transfected into the cells by calcium-phophateprecipitation (Ausubel, F. M., et al. (1993) Current Protocols inMolecular Biology, Greene and Wiley, USA).

Cell lines stably expressing hmGluRs are generated bylipofectin-mediated transfection (Gibco-BRL) of CHO-KL cells with hmGluRexpression plasmids and pSV2-Neo (Southern and Berg, 1982), a plasmidvector encoding the G-418 resistence gene. Cells are grown for 48 hoursprior to the addition of 1 mg/ml G-418 sulfate (Geneticin, Gibco).Medium is replaced every two to three days. Cells surviving the G-418selection are isolated and grown in the selection medium. 32 G-418resistant clonal cell lines are analyzed six to eight weeks after theinitial transfection for hmGluR protein expression by immunoreactivitywith the anti-hmGluR7 antibody (immunodetection, cf. 4.3, infra) andfunctional responses following agonist addition via cAMPradioimmunoassay (cf. 5.1, infra).

Likewise, the hmGluR expression constructs pBK-CMV-hmGluR4,pCMV-T7-hmGluR4, pCMV-T7-hmGluR7b and pCMV-T7-hmGluR7a are transientlyand stably expressed in mammalian cells (CV 1, CHO, HEK293, COS)according to standard procedures (Ausubel, F. M., et al. (1993) CurrentProtocols in Molecular Biology, Greene and Wiley, USA). The transfectedcells are analyzed for hmGluR expression by various assays:[³H]-glutamate binding studies, immunocytochemistry using hmGluR subtypespecific antibodies, and assays detecting a change in the intracellularconcentration of cAMP ([cAMP]).

4.3 Immunodetection of hmGluR Protein Expression with Subtype-SpecifichmGluR Antibodies

HmGluR protein expression is analyzed by immunocytochemistry withsubtype-specific hmGluR antibodies (see Example 7). 1 to 3 days aftertransfection cells are washed twice with phosphate buffered saline(PBS), fixed with PBS/4% paraformaldehyde for 10 min and washed withPBS. Cells are permeabilized with PBS/0.4% Triton X-100, followed bywashing with PBS/10 mM glycine, and PBS. Cells are blocked with PBSTB(1×PBS/0.1% Triton X-100/1% BSA) for 1 h and subsequently incubated withimmunopurified hmGluR antiserum (0.5-2.0 μg/ml in PBSTB) for 1 h. Afterthree washes with PBS, cells are incubated for 1 h with alkalineperoxidase conjugated goat anti-rabbit IgG (1:200 in PBSTB; JacksonImmuno Research). Cells are washed three times with PBS andimmunoreactivity is detected with 0.4 mg/ml naphtolphosphate (Biorad)/1mg/ml Fast Red (Biorad)/10 mM Levamisole (Sigma)/100 mM Tris/HCl pH8.8/100 mM NaCl/50 mM MgCl₂. The staining reaction is stopped after 15min by subsequent washing with PBS. 2 to 4 cell lines, each homogenouslyexpressing hmGluR4, hmGluR6 or hmGluR7, are identified byimmunostaining.

EXAMPLE 5

Use of Stable Cell Lines Expressing hmGluRs for the Screening ofModulators of Receptor Activity

Stable cell lines expressing hmGluR4, hmGluR6 and hmGluR7 are used toscreen for agonists, antagonists and allosteric modulators. Suchcompounds are identified by binding studies employing [³H]glutamateand/or measurement of changes in intracellular second messenger levels([cAMP], [Ca²⁺]).

5.1 cAMP Radioimmunoassay

Ligand binding and agonist-induced depression of forskolin stimulatedcAMP accumulation (changes in the intracellular cAMP concentration) areanalyzed by cAMP radioimmunoassay (Amersham). Cells are seeded in12-well plates at a density of 0.5-2.0×10⁵ cells per well and grown for2 to 4 days until a confluent layer of cells is obtained. Cells arewashed twice with PBS and incubated for 20 min in PBS containing 1 mM3-isobutyl-1-methylxanthine (IBMX). Cells are incubated with fresh PBScontaining 10 μM forskolin, 1 mM IBMX and a known hmGluR agonist for 20min. The agonistic effect is stopped and cAMP produced by the cells isreleased by adding 1 ml of ethanol-water-HCl mix (100 ml of ethanol, 50ml of water, 1 ml of 1 M HCl) after having aspirated the drug containingmedium. cAMP levels are determined by a cAMP radioimmunoassay involving[³H] cAMP (Amersham).

HmGluR subtypes 4, 6 and 7 are negatively coupled to adenylate cyclasewhen expressed in CHO cells. Agonist binding leads to an inhibition offorskolin induced cAMP accumulation. All subtypes are AP-4 sensitive,meaning that AP 4 has an agonistic effect in a concentration less than 1mM.

5.2 Measurement of Intracellular [Ca²⁺]

Cells transformed with one of the above expression plasmids are loadedwith a calcium sensitive fluorescent dye such as fura-2 or fluro-3. Toachieve this cells are plated in single wells, single wells containing acoverslip, or 96-well plates and grown for 1 to 5 days until a 50-100%confluent layer of cells is obtained. Wells are washed three times witha balance salt solution (BBS) and incubated for 1 h in BBS followed bythree additional washings with BBS. Then cells are incubated for 20 to60 min in a solution containing 50 μg fura-2-AM (or fluro3-AM)(Molecular Probes, Inc.) 4.99 ml BBS, 75 μl DMSO and 6.25 μg Pluronic(Molecular Probes, Inc). The cells are washed 3 times with BBScontaining 2 mg/ml bovine albumin followed by three washes in BBS. Afterallowing recovery of the cells for at least 10 min they are used formicrofluorometric measurements of [Ca²⁺].

Cells are transferred to an apparatus for fluometry such as an invertedmicroscope, a spectrofluometer of a fluorescence reader. Fluorescence ofthe calcium indicator (e.g. fura-2 or fluo-3) is induced by illuminationwith light of a wavelength covered by the excitation spectrum of the dye(fura-2: 340/380 nm, fluo-3 3 480 nm). An increase in intracellular freeclaciom ion concentration is monitored as an increase of fura-2 orfluo-3 fluorescence excited at 340 nm and 480 nm, respectively, or adecrease of fura-2 fluorescence excited at 380 nm.

As a positive control L-glutamate is applied at a concentrationcorresponding to its EC₅₀ value onto the cells, thereby inducing ameasurable increase in the intracellular calcium ion concentration. Atest compound is said to be an agonist if it induces a Ca²⁺ signalcomparable to that induced by glutamate. A test compound is said to bean antagonist if the glutamate induced calcium signal is smaller in thepresence of the test compound than in the absence of the test compound.

EXAMPLE 6

Chimeric hmGluR4, 6 and 7 Receptors

Intracellular domains of mGluR1, particularly the second intracellularloop (i2) and the C-terminal region, have been shown to be critical forbinding of G-proteins, which activate the phospholipase C/Ca²⁺ signalingpathway, without changing the pharmacological profile of the receptor(Pin et al., EMBO J. 13, 342-348, (1994)). Conventional PCR mutagenesistechniques are used to exchange intracellular domains of hmGluRs 4,6,and 7 with corresponding domains of hmGluR1. Stable CHO cell lines aregenerated with hmGluR4/1, 6/1 and 7/1 chimeric expression constructsallowing to analyze the influence of modulators of receptor activity(hmGluRs 4,6,7) using Ca²⁺-dependent assays. In the following, wedescribe the generation of a chimeric hmGluR7/1 receptor. Expressionconstructs with chimeric hmGluR4/1 and hmGluR6/1 are generated usinganalogous cloning and PCR techniques.

(i) The expression construct pCMV-hmGluR7b is digested with EagI,thereby releasing the complete cDNA insert. The cDNA is cloned into theNotI site of pBluescript-Not, a derivative of pBluescript II(Stratagene) where the polylinker sequences between the unique KpnI andNotI sites are deleted. The resulting clone is designated aspBluescript-Not-hmGluR7.

(ii) The transmembrane region of hmGluR1 is cloned by PCR using primersderived from Masu et al., 1991, supra. The oligonucleotide with thesequence

5′-TATCTTGAGTGGAGTGACATAG-3′ (SEQ ID NO:21)

(corresponding to nt 1753 to 1774 of the Masu sequence) is used as senseprimer. The antisense primer has the sequence

5′-ACTGCGGACGTTCCTCTCAGG-3′ (SEQ ID NO:22)

corresponding to nt 2524 to 2544 of the Masu sequence. The C-terminalend of splice variants 1a, 1b and 1c is cleaved by PCR using primersderived from Masu et al., 1991, Tanabe et al., 1992, supra, and Pin etal., 1992 (Proc. Natl. Acad. Sci, USA, 89, 10331-10335 (1992)),respectively. The oligonucleotide having the sequence

5′-AAACCTGAGAGGAACGTCCGCAG-3′ (SEQ ID NO:23)

(corresponding to nt 2521 to nt 2543 of the Masu sequence) is used assense primer. The oligonucleotides having the sequences

5′-CTACAGGGTGGAAGAGCTTTGCTT-3′ SEQ ID NO:24 corresponding to nt 3577 to3600 of the Masu sequence,

5′-TCAAAGCTGCGCATGTGCCGACGG-3′ SEQ ID NO: 25 corresponding to nt 2698 to2721 of the Tanabe sequence, and

5′-TCAATAGACAGTGTTTTGGCGGTC-3′ SEQ ID NO: 26 corresponding to nt 2671 to2694 of the Pin sequence are used as antisense primers for hmGluR1a, 1band 1c, respectively.

The PCR fragment is cloned into pBluescript II and sequenced completely.

(iii) A chimeric cDNA fragment wherein the i2-loop of hmGluR7a orhmGluR7b (nt 2035 to 2106 of SEQ IDs 11 and 13, respectively) isreplaced with the corresponding sequences of hmGluR1 is generated by PCR(as described in Pin et al., 1994, supra). The fragment is digested withSmaI and BglII which cut at unique restriction sites flanking thei2-loop. The chimeric SmaI/BglII fragment is exchanged for theSmaI/BglII fragments is pBluescript-Not-mGluR7.

(iv) Additional replacement of the C-terminal domain of hmGluR7b orhmGluR7a with the corresponding sequences of the above mentioned hmGluR1splice variants is achieved by using the unique restriction sites BglIIand SacII flanking the C-terminal end of hmGluR7.

(v) The resulting chimeric hmGluR7/hmGluR1 cDNAs are sequenced anddigested with EagI, thereby releasing the complete cDNAs frompBluescript-Not. For stable expression in CHO cells, the chimeric cDNAsare cloned into the unique NotI site of the mammalian expression vectorpCMV-T7-2.

EXAMPLE 7

Generation and Application of Anti-hmGluR Antibodies

Peptides corresponding to the deduced C-terminal amino acid sequences ofhmGluR4 and hmGluR7 are synthesized and coupled to ovalbumin orTentagel. Polyclonal antisera are raised in rabbits. Human mGluRspecific antibodies are purified from the antisera by immunoaffinitychromatography on peptide columns. The hmGluR specific antibodies arecharacterized by ELISA and immunoblotting withglutathione-S-transferase/hmGluR fusion proteins (produced in E. coli)or human brain extracts. Antibodies specific for hmGluR4 and hmGluR7,respectively, are used to detect hmGluR receptors in transfected cellsand to analyze the cellular and subcellular expression pattern of thehmGluR receptor proteins in tissue sections of human brain material.Antibodies are raised against different hmGluR-specific peptidesconsisting of 20 amino acids and fusion proteins expressed in E.coli.Peptides are synthesized by solid-phase synthesis, coupled to keyholelimpit hemocyanin (KLH) or ovalbumin with glutaraldehyde. PCR fragmentscontaining the entire putative intracellular C-terminal fragment ofhmGluRs are cloned as BamHI/EcoRI fragments into the E. coli expressionplasmid pGEX-2T (Guan and Dixon, Analytical Biochemistry 192, 262-267(1991)) generating glutathione-S-transferase(GST)/hmGluR fusion genes.E. coli DH5a cells (Gibco-BRL) carrying expression plasmids withGST/hmGluR fusion genes are grown overnight at 37° C. in LB medium/100mg/ml ampicillin. The cultures are diluted 1:30 in LB and grown for 2 hat 30° C. Expression of fusion proteins is induced by treatment with 0.1mM isopropyl-b-D-thiogalactopyranoside for 3 h at 30° C. Cells areharvested by centrifugation at 5,000×g. The fusion protein is isolatedusing glutathione affinity chromatography.

Deposition Data

The following plasmids were deposited with the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124Braunschweig on Sep. 13, 1993:

Plasmid cmR1; accession no. DSM 8549

Plasmid cmR2; accession no. DSM 8550

26 1 2736 DNA Homo sapiens CDS (1)..(2736) 1 atg cct ggg aag aga ggc ttgggc tgg tgg tgg gcc cgg ctg ccc ctt 48 Met Pro Gly Lys Arg Gly Leu GlyTrp Trp Trp Ala Arg Leu Pro Leu 1 5 10 15 tgc ctg ctc ctc agc ctt tacggc ccc tgg atg cct tcc tcc ctg gga 96 Cys Leu Leu Leu Ser Leu Tyr GlyPro Trp Met Pro Ser Ser Leu Gly 20 25 30 aag ccc aaa ggc cac cct cac atgaat tcc atc cgc ata gat ggg gac 144 Lys Pro Lys Gly His Pro His Met AsnSer Ile Arg Ile Asp Gly Asp 35 40 45 atc aca ctg gga ggc ctg ttc ccg gtgcat ggc cgg ggc tca gag ggc 192 Ile Thr Leu Gly Gly Leu Phe Pro Val HisGly Arg Gly Ser Glu Gly 50 55 60 aag ccc tgt gga gaa ctt aag aag gaa aagggc atc cac cgg ctg gag 240 Lys Pro Cys Gly Glu Leu Lys Lys Glu Lys GlyIle His Arg Leu Glu 65 70 75 80 gcc atg ctg ttc gcc ctg gat cgc atc aacaac gac ccg gac ctg ctg 288 Ala Met Leu Phe Ala Leu Asp Arg Ile Asn AsnAsp Pro Asp Leu Leu 85 90 95 cct aac atc acg ctg ggc gcc cgc att ctg gacacc tgc tcc agg gac 336 Pro Asn Ile Thr Leu Gly Ala Arg Ile Leu Asp ThrCys Ser Arg Asp 100 105 110 acc cat gcc ctc gag cag tcg ctg acc ttt gtgcag gcg ctc atc gag 384 Thr His Ala Leu Glu Gln Ser Leu Thr Phe Val GlnAla Leu Ile Glu 115 120 125 aag gat ggc aca gag gtc cgc tgt ggc agt ggcggc cca ccc atc atc 432 Lys Asp Gly Thr Glu Val Arg Cys Gly Ser Gly GlyPro Pro Ile Ile 130 135 140 acc aag cct gaa cgt gtg gtg ggt gtc atc ggtgct tca ggg agc tcg 480 Thr Lys Pro Glu Arg Val Val Gly Val Ile Gly AlaSer Gly Ser Ser 145 150 155 160 gtc tcc atc atg gtg gcc aac atc ctt cgcctc ttc aag ata ccc cag 528 Val Ser Ile Met Val Ala Asn Ile Leu Arg LeuPhe Lys Ile Pro Gln 165 170 175 atc agc tac gcc tcc aca gcg cca gac ctgagt gac aac agc cgc tac 576 Ile Ser Tyr Ala Ser Thr Ala Pro Asp Leu SerAsp Asn Ser Arg Tyr 180 185 190 gac ttc ttc tcc cgc gtg gtg ccc tcg gacacg tac cag gcc cag gcc 624 Asp Phe Phe Ser Arg Val Val Pro Ser Asp ThrTyr Gln Ala Gln Ala 195 200 205 atg gtg gac atc gtc cgt gcc ctc aag tggaac tat gtg tcc aca gtg 672 Met Val Asp Ile Val Arg Ala Leu Lys Trp AsnTyr Val Ser Thr Val 210 215 220 gcc tcg gag ggc agc tat ggt gag agc ggtgtg gag gcc ttc atc cag 720 Ala Ser Glu Gly Ser Tyr Gly Glu Ser Gly ValGlu Ala Phe Ile Gln 225 230 235 240 aag tcc cgt gag gac ggg ggc gtg tgcatc gcc cag tcg gtg aag ata 768 Lys Ser Arg Glu Asp Gly Gly Val Cys IleAla Gln Ser Val Lys Ile 245 250 255 cca cgg gag ccc aag gca ggc gag ttcgac aag atc atc cgc cgc ctc 816 Pro Arg Glu Pro Lys Ala Gly Glu Phe AspLys Ile Ile Arg Arg Leu 260 265 270 ctg gag act tcg aac gcc agg gca gtcatc atc ttt gcc aac gag gat 864 Leu Glu Thr Ser Asn Ala Arg Ala Val IleIle Phe Ala Asn Glu Asp 275 280 285 gac atc agg cgt gtg ctg gag gca gcacga agg gcc aac cag aca ggc 912 Asp Ile Arg Arg Val Leu Glu Ala Ala ArgArg Ala Asn Gln Thr Gly 290 295 300 cat ttc ttc tgg atg ggc tct gac agctgg ggc tcc aag att gca cct 960 His Phe Phe Trp Met Gly Ser Asp Ser TrpGly Ser Lys Ile Ala Pro 305 310 315 320 gtg ctg cac ctg gag gag gtg gctgag ggt gct gtc acg atc ctc ccc 1008 Val Leu His Leu Glu Glu Val Ala GluGly Ala Val Thr Ile Leu Pro 325 330 335 aag agg atg tcc gta cga ggc ttcgac cgc tac ttc tcc agc cgc acg 1056 Lys Arg Met Ser Val Arg Gly Phe AspArg Tyr Phe Ser Ser Arg Thr 340 345 350 ctg gac aac aac cgg cgc aac atctgg ttt gcc gag ttc tgg gag gac 1104 Leu Asp Asn Asn Arg Arg Asn Ile TrpPhe Ala Glu Phe Trp Glu Asp 355 360 365 aac ttc cac tgc aag ctg agc cgccac gcc ctc aag aag ggc agc cac 1152 Asn Phe His Cys Lys Leu Ser Arg HisAla Leu Lys Lys Gly Ser His 370 375 380 gtc aag aag tgc acc aac cgt gagcga att ggg cag gat tca gct tat 1200 Val Lys Lys Cys Thr Asn Arg Glu ArgIle Gly Gln Asp Ser Ala Tyr 385 390 395 400 gag cag gag ggg aag gtg cagttt gtg atc gat gcc gtg tac gcc atg 1248 Glu Gln Glu Gly Lys Val Gln PheVal Ile Asp Ala Val Tyr Ala Met 405 410 415 ggc cac gcg ctg cac gcc atgcac cgt gac ctg tgt ccc ggc cgc gtg 1296 Gly His Ala Leu His Ala Met HisArg Asp Leu Cys Pro Gly Arg Val 420 425 430 ggg ctc tgc ccg cgc atg gaccct gta gat ggc acc cag ctg ctt aag 1344 Gly Leu Cys Pro Arg Met Asp ProVal Asp Gly Thr Gln Leu Leu Lys 435 440 445 tac atc cga aac gtc aac ttctca ggc atc gca ggg aac cct gtg acc 1392 Tyr Ile Arg Asn Val Asn Phe SerGly Ile Ala Gly Asn Pro Val Thr 450 455 460 ttc aat gag aat gga gat gcgcct ggg cgc tat gac atc tac caa tac 1440 Phe Asn Glu Asn Gly Asp Ala ProGly Arg Tyr Asp Ile Tyr Gln Tyr 465 470 475 480 cag ctg cgc aac gat tctgcc gag tac aag gtc att ggc tcc tgg act 1488 Gln Leu Arg Asn Asp Ser AlaGlu Tyr Lys Val Ile Gly Ser Trp Thr 485 490 495 gac cac ctg cac ctt agaata gag cgg atg cac tgg ccg ggg agc ggg 1536 Asp His Leu His Leu Arg IleGlu Arg Met His Trp Pro Gly Ser Gly 500 505 510 cag cag ctg ccc cgc tccatc tgc agc ctg ccc tgc caa ccg ggt gag 1584 Gln Gln Leu Pro Arg Ser IleCys Ser Leu Pro Cys Gln Pro Gly Glu 515 520 525 cgg aag aag aca gtg aagggc atg cct tgc tgc tgg cac tgc gag cct 1632 Arg Lys Lys Thr Val Lys GlyMet Pro Cys Cys Trp His Cys Glu Pro 530 535 540 tgc aca ggg tac cag taccag gtg gac cgc tac acc tgt aag acg tgt 1680 Cys Thr Gly Tyr Gln Tyr GlnVal Asp Arg Tyr Thr Cys Lys Thr Cys 545 550 555 560 ccc tat gac atg cggccc aca gag aac cgc acg ggc tgc cgg ccc atc 1728 Pro Tyr Asp Met Arg ProThr Glu Asn Arg Thr Gly Cys Arg Pro Ile 565 570 575 ccc atc atc aag cttgag tgg ggc tcg ccc tgg gcc gtg ctg ccc ctc 1776 Pro Ile Ile Lys Leu GluTrp Gly Ser Pro Trp Ala Val Leu Pro Leu 580 585 590 ttc ctg gcc gtg gtgggc atc gct gcc acg ttg ttc gtg gtg atc acc 1824 Phe Leu Ala Val Val GlyIle Ala Ala Thr Leu Phe Val Val Ile Thr 595 600 605 ttt gtg cgc tac aacgac acg ccc atc gtc aag gcc tcg ggc cgt gaa 1872 Phe Val Arg Tyr Asn AspThr Pro Ile Val Lys Ala Ser Gly Arg Glu 610 615 620 ctg agc tac gtg ctgctg gca ggc atc ttc ctg tgc tat gcc acc acc 1920 Leu Ser Tyr Val Leu LeuAla Gly Ile Phe Leu Cys Tyr Ala Thr Thr 625 630 635 640 ttc ctc atg atcgct gag ccc gac ctt ggc acc tgc tcg ctg cgc cga 1968 Phe Leu Met Ile AlaGlu Pro Asp Leu Gly Thr Cys Ser Leu Arg Arg 645 650 655 atc ttc ctg ggacta ggg atg agc atc agc tat gca gcc ctg ctc acc 2016 Ile Phe Leu Gly LeuGly Met Ser Ile Ser Tyr Ala Ala Leu Leu Thr 660 665 670 aag acc aac cgcatc tac cgc atc ttc gag cag ggc aag cgc tcg gtc 2064 Lys Thr Asn Arg IleTyr Arg Ile Phe Glu Gln Gly Lys Arg Ser Val 675 680 685 agt gcc cca cgcttc atc agc ccc gcc tca cag ctg gcc atc acc ttc 2112 Ser Ala Pro Arg PheIle Ser Pro Ala Ser Gln Leu Ala Ile Thr Phe 690 695 700 agc ctc atc tcgctg cag ctg ctg ggc atc tgt gtg tgg ttt gtg gtg 2160 Ser Leu Ile Ser LeuGln Leu Leu Gly Ile Cys Val Trp Phe Val Val 705 710 715 720 gac ccc tcccac tcg gtg gtg gac ttc cag gac cag cgg aca ctc gac 2208 Asp Pro Ser HisSer Val Val Asp Phe Gln Asp Gln Arg Thr Leu Asp 725 730 735 ccc cgc ttcgcc agg ggt gtg ctc aag tgt gac atc tcg gac ctg tcg 2256 Pro Arg Phe AlaArg Gly Val Leu Lys Cys Asp Ile Ser Asp Leu Ser 740 745 750 ctc atc tgcctg ctg ggc tac agc atg ctg ctc atg gtc acg tgc acc 2304 Leu Ile Cys LeuLeu Gly Tyr Ser Met Leu Leu Met Val Thr Cys Thr 755 760 765 gtg tat gccatc aag aca cgc ggc gtg ccc gag acc ttc aat gag gcc 2352 Val Tyr Ala IleLys Thr Arg Gly Val Pro Glu Thr Phe Asn Glu Ala 770 775 780 aag ccc attggc ttc acc atg tac acc act tgc atc gtc tgg ctg gcc 2400 Lys Pro Ile GlyPhe Thr Met Tyr Thr Thr Cys Ile Val Trp Leu Ala 785 790 795 800 ttc atcccc atc ttc ttt ggc acc tcg cag tcg gcc gac aag ctg tac 2448 Phe Ile ProIle Phe Phe Gly Thr Ser Gln Ser Ala Asp Lys Leu Tyr 805 810 815 atc cagacg acg acg ctg acg gtc tcg gtg agt ctg agc gcc tcg gtg 2496 Ile Gln ThrThr Thr Leu Thr Val Ser Val Ser Leu Ser Ala Ser Val 820 825 830 tcc ctggga atg ctc tac atg ccc aaa gtc tac atc atc ctc ttc cac 2544 Ser Leu GlyMet Leu Tyr Met Pro Lys Val Tyr Ile Ile Leu Phe His 835 840 845 ccg gagcag aac gtg ccc aag cgc aag cgc agc ctc aaa gcc gtc gtt 2592 Pro Glu GlnAsn Val Pro Lys Arg Lys Arg Ser Leu Lys Ala Val Val 850 855 860 acg gcggcc acc atg tcc aac aag ttc acg cag aag ggc aac ttc cgg 2640 Thr Ala AlaThr Met Ser Asn Lys Phe Thr Gln Lys Gly Asn Phe Arg 865 870 875 880 cccaac gga gag gcc aag tct gag ctc tgc gag aac ctt gag gcc cca 2688 Pro AsnGly Glu Ala Lys Ser Glu Leu Cys Glu Asn Leu Glu Ala Pro 885 890 895 gcgctg gcc acc aaa cag act tac gtc act tac acc aac cat gca atc 2736 Ala LeuAla Thr Lys Gln Thr Tyr Val Thr Tyr Thr Asn His Ala Ile 900 905 910 2912 PRT Homo sapiens 2 Met Pro Gly Lys Arg Gly Leu Gly Trp Trp Trp AlaArg Leu Pro Leu 1 5 10 15 Cys Leu Leu Leu Ser Leu Tyr Gly Pro Trp MetPro Ser Ser Leu Gly 20 25 30 Lys Pro Lys Gly His Pro His Met Asn Ser IleArg Ile Asp Gly Asp 35 40 45 Ile Thr Leu Gly Gly Leu Phe Pro Val His GlyArg Gly Ser Glu Gly 50 55 60 Lys Pro Cys Gly Glu Leu Lys Lys Glu Lys GlyIle His Arg Leu Glu 65 70 75 80 Ala Met Leu Phe Ala Leu Asp Arg Ile AsnAsn Asp Pro Asp Leu Leu 85 90 95 Pro Asn Ile Thr Leu Gly Ala Arg Ile LeuAsp Thr Cys Ser Arg Asp 100 105 110 Thr His Ala Leu Glu Gln Ser Leu ThrPhe Val Gln Ala Leu Ile Glu 115 120 125 Lys Asp Gly Thr Glu Val Arg CysGly Ser Gly Gly Pro Pro Ile Ile 130 135 140 Thr Lys Pro Glu Arg Val ValGly Val Ile Gly Ala Ser Gly Ser Ser 145 150 155 160 Val Ser Ile Met ValAla Asn Ile Leu Arg Leu Phe Lys Ile Pro Gln 165 170 175 Ile Ser Tyr AlaSer Thr Ala Pro Asp Leu Ser Asp Asn Ser Arg Tyr 180 185 190 Asp Phe PheSer Arg Val Val Pro Ser Asp Thr Tyr Gln Ala Gln Ala 195 200 205 Met ValAsp Ile Val Arg Ala Leu Lys Trp Asn Tyr Val Ser Thr Val 210 215 220 AlaSer Glu Gly Ser Tyr Gly Glu Ser Gly Val Glu Ala Phe Ile Gln 225 230 235240 Lys Ser Arg Glu Asp Gly Gly Val Cys Ile Ala Gln Ser Val Lys Ile 245250 255 Pro Arg Glu Pro Lys Ala Gly Glu Phe Asp Lys Ile Ile Arg Arg Leu260 265 270 Leu Glu Thr Ser Asn Ala Arg Ala Val Ile Ile Phe Ala Asn GluAsp 275 280 285 Asp Ile Arg Arg Val Leu Glu Ala Ala Arg Arg Ala Asn GlnThr Gly 290 295 300 His Phe Phe Trp Met Gly Ser Asp Ser Trp Gly Ser LysIle Ala Pro 305 310 315 320 Val Leu His Leu Glu Glu Val Ala Glu Gly AlaVal Thr Ile Leu Pro 325 330 335 Lys Arg Met Ser Val Arg Gly Phe Asp ArgTyr Phe Ser Ser Arg Thr 340 345 350 Leu Asp Asn Asn Arg Arg Asn Ile TrpPhe Ala Glu Phe Trp Glu Asp 355 360 365 Asn Phe His Cys Lys Leu Ser ArgHis Ala Leu Lys Lys Gly Ser His 370 375 380 Val Lys Lys Cys Thr Asn ArgGlu Arg Ile Gly Gln Asp Ser Ala Tyr 385 390 395 400 Glu Gln Glu Gly LysVal Gln Phe Val Ile Asp Ala Val Tyr Ala Met 405 410 415 Gly His Ala LeuHis Ala Met His Arg Asp Leu Cys Pro Gly Arg Val 420 425 430 Gly Leu CysPro Arg Met Asp Pro Val Asp Gly Thr Gln Leu Leu Lys 435 440 445 Tyr IleArg Asn Val Asn Phe Ser Gly Ile Ala Gly Asn Pro Val Thr 450 455 460 PheAsn Glu Asn Gly Asp Ala Pro Gly Arg Tyr Asp Ile Tyr Gln Tyr 465 470 475480 Gln Leu Arg Asn Asp Ser Ala Glu Tyr Lys Val Ile Gly Ser Trp Thr 485490 495 Asp His Leu His Leu Arg Ile Glu Arg Met His Trp Pro Gly Ser Gly500 505 510 Gln Gln Leu Pro Arg Ser Ile Cys Ser Leu Pro Cys Gln Pro GlyGlu 515 520 525 Arg Lys Lys Thr Val Lys Gly Met Pro Cys Cys Trp His CysGlu Pro 530 535 540 Cys Thr Gly Tyr Gln Tyr Gln Val Asp Arg Tyr Thr CysLys Thr Cys 545 550 555 560 Pro Tyr Asp Met Arg Pro Thr Glu Asn Arg ThrGly Cys Arg Pro Ile 565 570 575 Pro Ile Ile Lys Leu Glu Trp Gly Ser ProTrp Ala Val Leu Pro Leu 580 585 590 Phe Leu Ala Val Val Gly Ile Ala AlaThr Leu Phe Val Val Ile Thr 595 600 605 Phe Val Arg Tyr Asn Asp Thr ProIle Val Lys Ala Ser Gly Arg Glu 610 615 620 Leu Ser Tyr Val Leu Leu AlaGly Ile Phe Leu Cys Tyr Ala Thr Thr 625 630 635 640 Phe Leu Met Ile AlaGlu Pro Asp Leu Gly Thr Cys Ser Leu Arg Arg 645 650 655 Ile Phe Leu GlyLeu Gly Met Ser Ile Ser Tyr Ala Ala Leu Leu Thr 660 665 670 Lys Thr AsnArg Ile Tyr Arg Ile Phe Glu Gln Gly Lys Arg Ser Val 675 680 685 Ser AlaPro Arg Phe Ile Ser Pro Ala Ser Gln Leu Ala Ile Thr Phe 690 695 700 SerLeu Ile Ser Leu Gln Leu Leu Gly Ile Cys Val Trp Phe Val Val 705 710 715720 Asp Pro Ser His Ser Val Val Asp Phe Gln Asp Gln Arg Thr Leu Asp 725730 735 Pro Arg Phe Ala Arg Gly Val Leu Lys Cys Asp Ile Ser Asp Leu Ser740 745 750 Leu Ile Cys Leu Leu Gly Tyr Ser Met Leu Leu Met Val Thr CysThr 755 760 765 Val Tyr Ala Ile Lys Thr Arg Gly Val Pro Glu Thr Phe AsnGlu Ala 770 775 780 Lys Pro Ile Gly Phe Thr Met Tyr Thr Thr Cys Ile ValTrp Leu Ala 785 790 795 800 Phe Ile Pro Ile Phe Phe Gly Thr Ser Gln SerAla Asp Lys Leu Tyr 805 810 815 Ile Gln Thr Thr Thr Leu Thr Val Ser ValSer Leu Ser Ala Ser Val 820 825 830 Ser Leu Gly Met Leu Tyr Met Pro LysVal Tyr Ile Ile Leu Phe His 835 840 845 Pro Glu Gln Asn Val Pro Lys ArgLys Arg Ser Leu Lys Ala Val Val 850 855 860 Thr Ala Ala Thr Met Ser AsnLys Phe Thr Gln Lys Gly Asn Phe Arg 865 870 875 880 Pro Asn Gly Glu AlaLys Ser Glu Leu Cys Glu Asn Leu Glu Ala Pro 885 890 895 Ala Leu Ala ThrLys Gln Thr Tyr Val Thr Tyr Thr Asn His Ala Ile 900 905 910 3 3804 DNAHomo sapiens CDS (1)..(2604) unsure (3325)..(3495) nucleotidesdesignated as n could be a or g or c or t/u 3 ccc gta cac gcc aag ggtccc agc gga gtg ccc tgc ggc gac atc aag 48 Pro Val His Ala Lys Gly ProSer Gly Val Pro Cys Gly Asp Ile Lys 1 5 10 15 agg gaa aac ggg atc cacagg ctg gaa gcg atg ctc tac gcc ctg gac 96 Arg Glu Asn Gly Ile His ArgLeu Glu Ala Met Leu Tyr Ala Leu Asp 20 25 30 cag atc aac agt gat ccc aaccta ctg ccc aac gtg acg ctg ggc gcg 144 Gln Ile Asn Ser Asp Pro Asn LeuLeu Pro Asn Val Thr Leu Gly Ala 35 40 45 cgg atc ctg gac act tgt tcc agggac act tac gcg ctc gaa cag tcg 192 Arg Ile Leu Asp Thr Cys Ser Arg AspThr Tyr Ala Leu Glu Gln Ser 50 55 60 ctt act ttc gtc cag gcg ctc atc cagaag gac acc tcc gac gtg cgc 240 Leu Thr Phe Val Gln Ala Leu Ile Gln LysAsp Thr Ser Asp Val Arg 65 70 75 80 tgc acc aac ggc gaa ccg ccg gtt ttcgtc aag ccg gag aaa gta gtt 288 Cys Thr Asn Gly Glu Pro Pro Val Phe ValLys Pro Glu Lys Val Val 85 90 95 gga gtg att ggg gct tcg ggg agt tcg gtctcc atc atg gta gcc aac 336 Gly Val Ile Gly Ala Ser Gly Ser Ser Val SerIle Met Val Ala Asn 100 105 110 atc ctg agg ctc ttc cag atc ccc cag attagt tat gca tca acg gca 384 Ile Leu Arg Leu Phe Gln Ile Pro Gln Ile SerTyr Ala Ser Thr Ala 115 120 125 ccc gag cta agt gat gac cgg cgc tat gacttc ttc tct cgc gtg gtg 432 Pro Glu Leu Ser Asp Asp Arg Arg Tyr Asp PhePhe Ser Arg Val Val 130 135 140 cca ccc gat tcc ttc caa gcc cag gcc atggta gac att gta aag gcc 480 Pro Pro Asp Ser Phe Gln Ala Gln Ala Met ValAsp Ile Val Lys Ala 145 150 155 160 cta ggc tgg aat tat gtg tct acc ctcgca tcg gaa gga agt tat gga 528 Leu Gly Trp Asn Tyr Val Ser Thr Leu AlaSer Glu Gly Ser Tyr Gly 165 170 175 gag aaa ggt gtg gag tcc ttc acg cagatt tcc aaa gag gca ggt gga 576 Glu Lys Gly Val Glu Ser Phe Thr Gln IleSer Lys Glu Ala Gly Gly 180 185 190 ctc tgc att gcc cag tcc gtg aga atcccc cag gaa cgc aaa gac agg 624 Leu Cys Ile Ala Gln Ser Val Arg Ile ProGln Glu Arg Lys Asp Arg 195 200 205 acc att gac ttt gat aga att atc aaacag ctc ctg gac acc ccc aac 672 Thr Ile Asp Phe Asp Arg Ile Ile Lys GlnLeu Leu Asp Thr Pro Asn 210 215 220 tcc agg gcc gtc gtg att ttt gcc aacgat gag gat ata aag cag atc 720 Ser Arg Ala Val Val Ile Phe Ala Asn AspGlu Asp Ile Lys Gln Ile 225 230 235 240 ctt gca gca gcc aaa aga gct gaccaa gtt ggc cat ttt ctt tgg gtg 768 Leu Ala Ala Ala Lys Arg Ala Asp GlnVal Gly His Phe Leu Trp Val 245 250 255 gga tca gac agc tgg gga tcc aaaata aac cca ctg cac cag cat gaa 816 Gly Ser Asp Ser Trp Gly Ser Lys IleAsn Pro Leu His Gln His Glu 260 265 270 gat atc gca gaa ggg gcc atc accatt cag ccc aag cga gcc acg gtg 864 Asp Ile Ala Glu Gly Ala Ile Thr IleGln Pro Lys Arg Ala Thr Val 275 280 285 gaa ggg ttt gat gcc tac ttt acgtcc cgt aca ctt gaa aac aac aga 912 Glu Gly Phe Asp Ala Tyr Phe Thr SerArg Thr Leu Glu Asn Asn Arg 290 295 300 aga aat gta tgg ttt gcc gaa tactgg gag gaa aac ttc aac tgc aag 960 Arg Asn Val Trp Phe Ala Glu Tyr TrpGlu Glu Asn Phe Asn Cys Lys 305 310 315 320 ttg acg att agt ggg tca aaaaaa gaa gac aca gat cgc aaa tgc aca 1008 Leu Thr Ile Ser Gly Ser Lys LysGlu Asp Thr Asp Arg Lys Cys Thr 325 330 335 gga cag gag aga att gga aaagat tcc aac tat gag cag gag ggt aaa 1056 Gly Gln Glu Arg Ile Gly Lys AspSer Asn Tyr Glu Gln Glu Gly Lys 340 345 350 gtc cag ttc gtg att gac gcagtc tat gct atg gct cac gcc ctt cac 1104 Val Gln Phe Val Ile Asp Ala ValTyr Ala Met Ala His Ala Leu His 355 360 365 cac atg aac aag gat ctc tgtgct gac tac cgg ggt gtc tgc cca gag 1152 His Met Asn Lys Asp Leu Cys AlaAsp Tyr Arg Gly Val Cys Pro Glu 370 375 380 atg gag caa gct gga ggc aagaag ttg ctg aag tat ata cgc aat gtt 1200 Met Glu Gln Ala Gly Gly Lys LysLeu Leu Lys Tyr Ile Arg Asn Val 385 390 395 400 aat ttc aat ggt agt gctggc act cca gtg atg ttt aac aag aac ggg 1248 Asn Phe Asn Gly Ser Ala GlyThr Pro Val Met Phe Asn Lys Asn Gly 405 410 415 gat gca cct ggg cgt tatgac atc ttt cag tac cag acc aca aac acc 1296 Asp Ala Pro Gly Arg Tyr AspIle Phe Gln Tyr Gln Thr Thr Asn Thr 420 425 430 agc aac ccg ggt tac cgtctg atc ggg cag tgg aca gac gaa ctt cag 1344 Ser Asn Pro Gly Tyr Arg LeuIle Gly Gln Trp Thr Asp Glu Leu Gln 435 440 445 ctc aat ata gaa gac atgcag tgg ggt aaa gga gtc cga gag ata ccc 1392 Leu Asn Ile Glu Asp Met GlnTrp Gly Lys Gly Val Arg Glu Ile Pro 450 455 460 gcc tca gtg tgc aca ctacca tgt aag cca gga cag aga aag aag aca 1440 Ala Ser Val Cys Thr Leu ProCys Lys Pro Gly Gln Arg Lys Lys Thr 465 470 475 480 cag aaa gga act ccttgc tgt tgg acc tgt gag cct tgc gat ggt tac 1488 Gln Lys Gly Thr Pro CysCys Trp Thr Cys Glu Pro Cys Asp Gly Tyr 485 490 495 cag tac cag ttt gatgag atg aca tgc cag cat tgc ccc tat gac cag 1536 Gln Tyr Gln Phe Asp GluMet Thr Cys Gln His Cys Pro Tyr Asp Gln 500 505 510 agg ccc aat gaa aatcga acc gga tgc cag gat att ccc atc atc aaa 1584 Arg Pro Asn Glu Asn ArgThr Gly Cys Gln Asp Ile Pro Ile Ile Lys 515 520 525 ctg gag tgg cac tccccc tgg gct gtg att cct gtc ttc ctg gca atg 1632 Leu Glu Trp His Ser ProTrp Ala Val Ile Pro Val Phe Leu Ala Met 530 535 540 ttg ggg atc att gccacc atc ttt gtc atg gcc act ttc atc cgc tac 1680 Leu Gly Ile Ile Ala ThrIle Phe Val Met Ala Thr Phe Ile Arg Tyr 545 550 555 560 aat gac acg cccatt gtc cgg gca tct ggg cgg gaa ctc agc tat gtt 1728 Asn Asp Thr Pro IleVal Arg Ala Ser Gly Arg Glu Leu Ser Tyr Val 565 570 575 ctt ttg acg ggcatc ttt ctt tgc tac atc atc act ttc ctg atg att 1776 Leu Leu Thr Gly IlePhe Leu Cys Tyr Ile Ile Thr Phe Leu Met Ile 580 585 590 gcc aaa cca gatgtg gca gtg tgt tct ttc cgg cga gtt ttc ttg ggc 1824 Ala Lys Pro Asp ValAla Val Cys Ser Phe Arg Arg Val Phe Leu Gly 595 600 605 ttg ggt atg tgcatc agt tat gca gcc ctc ttg acg aaa aca aat cgg 1872 Leu Gly Met Cys IleSer Tyr Ala Ala Leu Leu Thr Lys Thr Asn Arg 610 615 620 att tat cgc atattt gag cag ggc aag aaa tca gta aca gct ccc aga 1920 Ile Tyr Arg Ile PheGlu Gln Gly Lys Lys Ser Val Thr Ala Pro Arg 625 630 635 640 ctc ata agccca aca tca caa ctg gca atc act tcc agt tta ata tca 1968 Leu Ile Ser ProThr Ser Gln Leu Ala Ile Thr Ser Ser Leu Ile Ser 645 650 655 gtt cag cttcta ggg gtg ttc att tgg ttt ggt gtt gat cca ccc aac 2016 Val Gln Leu LeuGly Val Phe Ile Trp Phe Gly Val Asp Pro Pro Asn 660 665 670 atc atc atagac tac gat gaa cac aag aca atg aac cct gag caa gcc 2064 Ile Ile Ile AspTyr Asp Glu His Lys Thr Met Asn Pro Glu Gln Ala 675 680 685 aga ggg gttctc aag tgt gac att aca gat ctc caa atc att tgc tcc 2112 Arg Gly Val LeuLys Cys Asp Ile Thr Asp Leu Gln Ile Ile Cys Ser 690 695 700 ttg gga tatagc att ctt ctc atg gtc aca tgt act gtg tat gcc atc 2160 Leu Gly Tyr SerIle Leu Leu Met Val Thr Cys Thr Val Tyr Ala Ile 705 710 715 720 aag actcgg ggt gta ccc gag aat ttt aac gaa gcc aag ccc att gga 2208 Lys Thr ArgGly Val Pro Glu Asn Phe Asn Glu Ala Lys Pro Ile Gly 725 730 735 ttc actatg tac acg aca tgt ata gta tgg ctt gcc ttc att cca att 2256 Phe Thr MetTyr Thr Thr Cys Ile Val Trp Leu Ala Phe Ile Pro Ile 740 745 750 ttt tttggc acc gct caa tca gcg gaa aag ctc tac ata caa act acc 2304 Phe Phe GlyThr Ala Gln Ser Ala Glu Lys Leu Tyr Ile Gln Thr Thr 755 760 765 acg cttaca atc tcc atg aac cta agt gca tca gtg gcg ctg ggg atg 2352 Thr Leu ThrIle Ser Met Asn Leu Ser Ala Ser Val Ala Leu Gly Met 770 775 780 cta tacatg ccg aaa gtg tac atc atc att ttc cac cct gaa ctc aat 2400 Leu Tyr MetPro Lys Val Tyr Ile Ile Ile Phe His Pro Glu Leu Asn 785 790 795 800 gtccag aaa cgg aag cga agc ttc aag gcg gta gtc aca gca gcc acc 2448 Val GlnLys Arg Lys Arg Ser Phe Lys Ala Val Val Thr Ala Ala Thr 805 810 815 atgtca tcg agg ctg tca cac aaa ccc agt gac aga ccc aac ggt gag 2496 Met SerSer Arg Leu Ser His Lys Pro Ser Asp Arg Pro Asn Gly Glu 820 825 830 gcaaag acc gag ctc tgt gaa aac gta gac cca aac aac tgt ata cca 2544 Ala LysThr Glu Leu Cys Glu Asn Val Asp Pro Asn Asn Cys Ile Pro 835 840 845 ccagta aga aag agt gta caa aag tct gtt act tgg tac act atc cca 2592 Pro ValArg Lys Ser Val Gln Lys Ser Val Thr Trp Tyr Thr Ile Pro 850 855 860 ccaaca gta tag cttttgactg ctttcccaaa ggccctgctg caaaaaagaa 2644 Pro Thr Val865 gtatgtcagt tataataacc tggttatcta acctgttcca ttccatggaa ccatggagga2704 ggaagaccct cagttatttt gtcacccaac ctggcatagg actctttggt cctacccgct2764 tcccatcacc ggaggagctt ccccggccgg gagaccagtg ttagaggatc caagcgacct2824 aaacagctgc tttatgaaat atccttactt tatctgggct taataagtca ctgacatcag2884 cactgccaac ttggctgcaa ttgtggacct tccctaccaa agggagtgtt gaaactcaag2944 tcccgccccg gctctttaga atggaccact gagagccaca ggaccgtttt ggggctgacc3004 tgtcttatta cgtatgtact tctaggttgc aaggttttga aattttctgt acagtttgtg3064 aggacctttg cactttgcca tctgatgtcg tacctcggtt cactgtttgt tttcgaatgc3124 cttgttttca tagagcccta ttctctcaga cggtggaata tttggaaaaa ttttaaaaca3184 attaaaattt taaagcaatc ttggcagact aaaacaagta catctgtaca tgactgtata3244 attacgttat agtaccactg cacatcatgt tttttttttt aagacaaaaa agatgtttaa3304 agaccaaaaa ctgtgctgag naagtatgcc ccacctatct ttngnatatg ataggttaca3364 taaaaggaag gtattggctg aactgnatag aggtcttgat ctttggaatg catgccagta3424 atgtatttac agtacatgtt tattatgttc aatatttgta tttgtgttct cttttgttat3484 ttttaattag ngtatatgaa tattttgcaa taattttaat aattattaag ctgtttgaag3544 gaaagaatat ggatttttca tgtcttgagg ttttgttcat gccccctttg actgatcagt3604 gtgataagga ctttaggaaa aaaagcatgt atgtttttta ctgtttgtaa taagtacttt3664 cgttaatctt gctgcttatg tgccaattta gtggaaaaga acaacccttg ctgaaaaatt3724 ccctctttcc attctctttc aattctgtga tattgtccaa gaatgtatca ataaaatact3784 ttggttaact ttaaaaaaaa 3804 4 867 PRT Homo sapiens 4 Pro Val His AlaLys Gly Pro Ser Gly Val Pro Cys Gly Asp Ile Lys 1 5 10 15 Arg Glu AsnGly Ile His Arg Leu Glu Ala Met Leu Tyr Ala Leu Asp 20 25 30 Gln Ile AsnSer Asp Pro Asn Leu Leu Pro Asn Val Thr Leu Gly Ala 35 40 45 Arg Ile LeuAsp Thr Cys Ser Arg Asp Thr Tyr Ala Leu Glu Gln Ser 50 55 60 Leu Thr PheVal Gln Ala Leu Ile Gln Lys Asp Thr Ser Asp Val Arg 65 70 75 80 Cys ThrAsn Gly Glu Pro Pro Val Phe Val Lys Pro Glu Lys Val Val 85 90 95 Gly ValIle Gly Ala Ser Gly Ser Ser Val Ser Ile Met Val Ala Asn 100 105 110 IleLeu Arg Leu Phe Gln Ile Pro Gln Ile Ser Tyr Ala Ser Thr Ala 115 120 125Pro Glu Leu Ser Asp Asp Arg Arg Tyr Asp Phe Phe Ser Arg Val Val 130 135140 Pro Pro Asp Ser Phe Gln Ala Gln Ala Met Val Asp Ile Val Lys Ala 145150 155 160 Leu Gly Trp Asn Tyr Val Ser Thr Leu Ala Ser Glu Gly Ser TyrGly 165 170 175 Glu Lys Gly Val Glu Ser Phe Thr Gln Ile Ser Lys Glu AlaGly Gly 180 185 190 Leu Cys Ile Ala Gln Ser Val Arg Ile Pro Gln Glu ArgLys Asp Arg 195 200 205 Thr Ile Asp Phe Asp Arg Ile Ile Lys Gln Leu LeuAsp Thr Pro Asn 210 215 220 Ser Arg Ala Val Val Ile Phe Ala Asn Asp GluAsp Ile Lys Gln Ile 225 230 235 240 Leu Ala Ala Ala Lys Arg Ala Asp GlnVal Gly His Phe Leu Trp Val 245 250 255 Gly Ser Asp Ser Trp Gly Ser LysIle Asn Pro Leu His Gln His Glu 260 265 270 Asp Ile Ala Glu Gly Ala IleThr Ile Gln Pro Lys Arg Ala Thr Val 275 280 285 Glu Gly Phe Asp Ala TyrPhe Thr Ser Arg Thr Leu Glu Asn Asn Arg 290 295 300 Arg Asn Val Trp PheAla Glu Tyr Trp Glu Glu Asn Phe Asn Cys Lys 305 310 315 320 Leu Thr IleSer Gly Ser Lys Lys Glu Asp Thr Asp Arg Lys Cys Thr 325 330 335 Gly GlnGlu Arg Ile Gly Lys Asp Ser Asn Tyr Glu Gln Glu Gly Lys 340 345 350 ValGln Phe Val Ile Asp Ala Val Tyr Ala Met Ala His Ala Leu His 355 360 365His Met Asn Lys Asp Leu Cys Ala Asp Tyr Arg Gly Val Cys Pro Glu 370 375380 Met Glu Gln Ala Gly Gly Lys Lys Leu Leu Lys Tyr Ile Arg Asn Val 385390 395 400 Asn Phe Asn Gly Ser Ala Gly Thr Pro Val Met Phe Asn Lys AsnGly 405 410 415 Asp Ala Pro Gly Arg Tyr Asp Ile Phe Gln Tyr Gln Thr ThrAsn Thr 420 425 430 Ser Asn Pro Gly Tyr Arg Leu Ile Gly Gln Trp Thr AspGlu Leu Gln 435 440 445 Leu Asn Ile Glu Asp Met Gln Trp Gly Lys Gly ValArg Glu Ile Pro 450 455 460 Ala Ser Val Cys Thr Leu Pro Cys Lys Pro GlyGln Arg Lys Lys Thr 465 470 475 480 Gln Lys Gly Thr Pro Cys Cys Trp ThrCys Glu Pro Cys Asp Gly Tyr 485 490 495 Gln Tyr Gln Phe Asp Glu Met ThrCys Gln His Cys Pro Tyr Asp Gln 500 505 510 Arg Pro Asn Glu Asn Arg ThrGly Cys Gln Asp Ile Pro Ile Ile Lys 515 520 525 Leu Glu Trp His Ser ProTrp Ala Val Ile Pro Val Phe Leu Ala Met 530 535 540 Leu Gly Ile Ile AlaThr Ile Phe Val Met Ala Thr Phe Ile Arg Tyr 545 550 555 560 Asn Asp ThrPro Ile Val Arg Ala Ser Gly Arg Glu Leu Ser Tyr Val 565 570 575 Leu LeuThr Gly Ile Phe Leu Cys Tyr Ile Ile Thr Phe Leu Met Ile 580 585 590 AlaLys Pro Asp Val Ala Val Cys Ser Phe Arg Arg Val Phe Leu Gly 595 600 605Leu Gly Met Cys Ile Ser Tyr Ala Ala Leu Leu Thr Lys Thr Asn Arg 610 615620 Ile Tyr Arg Ile Phe Glu Gln Gly Lys Lys Ser Val Thr Ala Pro Arg 625630 635 640 Leu Ile Ser Pro Thr Ser Gln Leu Ala Ile Thr Ser Ser Leu IleSer 645 650 655 Val Gln Leu Leu Gly Val Phe Ile Trp Phe Gly Val Asp ProPro Asn 660 665 670 Ile Ile Ile Asp Tyr Asp Glu His Lys Thr Met Asn ProGlu Gln Ala 675 680 685 Arg Gly Val Leu Lys Cys Asp Ile Thr Asp Leu GlnIle Ile Cys Ser 690 695 700 Leu Gly Tyr Ser Ile Leu Leu Met Val Thr CysThr Val Tyr Ala Ile 705 710 715 720 Lys Thr Arg Gly Val Pro Glu Asn PheAsn Glu Ala Lys Pro Ile Gly 725 730 735 Phe Thr Met Tyr Thr Thr Cys IleVal Trp Leu Ala Phe Ile Pro Ile 740 745 750 Phe Phe Gly Thr Ala Gln SerAla Glu Lys Leu Tyr Ile Gln Thr Thr 755 760 765 Thr Leu Thr Ile Ser MetAsn Leu Ser Ala Ser Val Ala Leu Gly Met 770 775 780 Leu Tyr Met Pro LysVal Tyr Ile Ile Ile Phe His Pro Glu Leu Asn 785 790 795 800 Val Gln LysArg Lys Arg Ser Phe Lys Ala Val Val Thr Ala Ala Thr 805 810 815 Met SerSer Arg Leu Ser His Lys Pro Ser Asp Arg Pro Asn Gly Glu 820 825 830 AlaLys Thr Glu Leu Cys Glu Asn Val Asp Pro Asn Asn Cys Ile Pro 835 840 845Pro Val Arg Lys Ser Val Gln Lys Ser Val Thr Trp Tyr Thr Ile Pro 850 855860 Pro Thr Val 865 5 1399 DNA Homo sapiens CDS (1)..(270) unsure(920)..(1090) Nucleotides designated as n could be a or g or c or t/u 5atc tcc atg aac cta agt gca tca gtg gcg ctg ggg atg cta tac atg 48 IleSer Met Asn Leu Ser Ala Ser Val Ala Leu Gly Met Leu Tyr Met 1 5 10 15ccg aaa gtg tac atc atc att ttc cac cct gaa ctc aat gtc cag aaa 96 ProLys Val Tyr Ile Ile Ile Phe His Pro Glu Leu Asn Val Gln Lys 20 25 30 cggaag cga agc ttc aag gcg gta gtc aca gca gcc acc atg tca tcg 144 Arg LysArg Ser Phe Lys Ala Val Val Thr Ala Ala Thr Met Ser Ser 35 40 45 agg ctgtca cac aaa ccc agt gac aga ccc aac ggt gag gca aag acc 192 Arg Leu SerHis Lys Pro Ser Asp Arg Pro Asn Gly Glu Ala Lys Thr 50 55 60 gag ctc tgtgaa aac gta gac cca aac agc cct gct gca aaa aag aag 240 Glu Leu Cys GluAsn Val Asp Pro Asn Ser Pro Ala Ala Lys Lys Lys 65 70 75 80 tat gtc agttat aat aac ctg gtt atc taa cctgttccat tccatggaac 290 Tyr Val Ser TyrAsn Asn Leu Val Ile 85 catggaggag gaagaccctc agttattttg tcacccaacctggcatagga ctctttggtc 350 ctacccgctt cccatcaccg gaggagcttc cccggccgggagaccagtgt tagaggatcc 410 aagcgaccta aacagctgct ttatgaaata tccttactttatctgggctt aataagtcac 470 tgacatcagc actgccaact tggctgcaat tgtggaccttccctaccaaa gggagtgttg 530 aaactcaagt cccgccccgg ctctttagaa tggaccactgagagccacag gaccgttttg 590 gggctgacct gtcttattac gtatgtactt ctaggttgcaaggttttgaa attttctgta 650 cagtttgtga ggacctttgc actttgccat ctgatgtcgtacctcggttc actgtttgtt 710 ttcgaatgcc ttgttttcat agagccctat tctctcagacggtggaatat ttggaaaaat 770 tttaaaacaa ttaaaatttt aaagcaatct tggcagactaaaacaagtac atctgtacat 830 gactgtataa ttacgttata gtaccactgc acatcatgttttttttttta agacaaaaaa 890 gatgtttaaa gaccaaaaac tgtgctgagn aagtatgccccacctatctt tngnatatga 950 taggttacat aaaaggaagg tattggctga actgnatagaggtcttgatc tttggaatgc 1010 atgccagtaa tgtatttaca gtacatgttt attatgttcaatatttgtat ttgtgttctc 1070 ttttgttatt tttaattagn gtatatgaat attttgcaataattttaata attattaagc 1130 tgtttgaagg aaagaatatg gatttttcat gtcttgaggttttgttcatg ccccctttga 1190 ctgatcagtg tgataaggac tttaggaaaa aaagcatgtatgttttttac tgtttgtaat 1250 aagtactttc gttaatcttg ctgcttatgt gccaatttagtggaaaagaa caacccttgc 1310 tgaaaaattc cctctttcca ttctctttca attctgtgatattgtccaag aatgtatcaa 1370 taaaatactt tggttaactt taaaaaaaa 1399 6 89 PRTHomo sapiens 6 Ile Ser Met Asn Leu Ser Ala Ser Val Ala Leu Gly Met LeuTyr Met 1 5 10 15 Pro Lys Val Tyr Ile Ile Ile Phe His Pro Glu Leu AsnVal Gln Lys 20 25 30 Arg Lys Arg Ser Phe Lys Ala Val Val Thr Ala Ala ThrMet Ser Ser 35 40 45 Arg Leu Ser His Lys Pro Ser Asp Arg Pro Asn Gly GluAla Lys Thr 50 55 60 Glu Leu Cys Glu Asn Val Asp Pro Asn Ser Pro Ala AlaLys Lys Lys 65 70 75 80 Tyr Val Ser Tyr Asn Asn Leu Val Ile 85 7 1588DNA Homo sapiens CDS (2)..(1447) 7 g aac aag gat ctc tgt gct gac tac cggggt gtc tgc cca gag atg gag 49 Asn Lys Asp Leu Cys Ala Asp Tyr Arg GlyVal Cys Pro Glu Met Glu 1 5 10 15 caa gct gga ggc aag aag ttg ctg aagtat ata cgc aat gtt aat ttc 97 Gln Ala Gly Gly Lys Lys Leu Leu Lys TyrIle Arg Asn Val Asn Phe 20 25 30 aat ggt agt gct ggc act cca gtg atg tttaac aag aac ggg gat gca 145 Asn Gly Ser Ala Gly Thr Pro Val Met Phe AsnLys Asn Gly Asp Ala 35 40 45 cct ggg cgt tat gac atc ttt cag tac cag accaca aac acc agc aac 193 Pro Gly Arg Tyr Asp Ile Phe Gln Tyr Gln Thr ThrAsn Thr Ser Asn 50 55 60 ccg ggt tac cgt ctg atc ggg cag tgg aca gac gaactt cag ctc aat 241 Pro Gly Tyr Arg Leu Ile Gly Gln Trp Thr Asp Glu LeuGln Leu Asn 65 70 75 80 ata gaa gac atg cag tgg ggt aaa gga gtc cga gagata ccc gcc tca 289 Ile Glu Asp Met Gln Trp Gly Lys Gly Val Arg Glu IlePro Ala Ser 85 90 95 gtg tgc aca cta cca tgt aag cca gga cag aga aag aagaca cag aaa 337 Val Cys Thr Leu Pro Cys Lys Pro Gly Gln Arg Lys Lys ThrGln Lys 100 105 110 gga act cct tgc tgt tgg acc tgt gag cct tgc gat ggttac cag tac 385 Gly Thr Pro Cys Cys Trp Thr Cys Glu Pro Cys Asp Gly TyrGln Tyr 115 120 125 cag ttt gat gag atg aca tgc cag cat tgc ccc tat gaccag agg ccc 433 Gln Phe Asp Glu Met Thr Cys Gln His Cys Pro Tyr Asp GlnArg Pro 130 135 140 aat gaa aat cga acc gga tgc cag gat att ccc atc atcaaa ctg gag 481 Asn Glu Asn Arg Thr Gly Cys Gln Asp Ile Pro Ile Ile LysLeu Glu 145 150 155 160 tgg cac tcc ccc tgg gct gtg att cct gtc ttc ctggca atg ttg ggg 529 Trp His Ser Pro Trp Ala Val Ile Pro Val Phe Leu AlaMet Leu Gly 165 170 175 atc att gcc acc atc ttt gtc atg gcc act ttc atccgc tac aat gac 577 Ile Ile Ala Thr Ile Phe Val Met Ala Thr Phe Ile ArgTyr Asn Asp 180 185 190 acg ccc att gtc cgg gca tct ggg cgg gaa ctc agctat gtt ctt ttg 625 Thr Pro Ile Val Arg Ala Ser Gly Arg Glu Leu Ser TyrVal Leu Leu 195 200 205 acg ggc atc ttt ctt tgc tac atc atc act ttc ctgatg att gcc aaa 673 Thr Gly Ile Phe Leu Cys Tyr Ile Ile Thr Phe Leu MetIle Ala Lys 210 215 220 cca gat gtg gca gtg tgt tct ttc cgg cga gtt ttcttg ggc ttg ggt 721 Pro Asp Val Ala Val Cys Ser Phe Arg Arg Val Phe LeuGly Leu Gly 225 230 235 240 atg tgc atc agt tat gca gcc ctc ttg acg aaaaca aat cgg att tat 769 Met Cys Ile Ser Tyr Ala Ala Leu Leu Thr Lys ThrAsn Arg Ile Tyr 245 250 255 cgc ata ttt gag cag ggc aag aaa tca gta acagct ccc aga ctc ata 817 Arg Ile Phe Glu Gln Gly Lys Lys Ser Val Thr AlaPro Arg Leu Ile 260 265 270 agc cca aca tca caa ctg gca atc act tcc agttta ata tca gtt cag 865 Ser Pro Thr Ser Gln Leu Ala Ile Thr Ser Ser LeuIle Ser Val Gln 275 280 285 ctt cta ggg gtg ttc att tgg ttt ggt gtt gatcca ccc aac atc atc 913 Leu Leu Gly Val Phe Ile Trp Phe Gly Val Asp ProPro Asn Ile Ile 290 295 300 ata gac tac gat gaa cac aag aca atg aac cctgag caa gcc aga ggg 961 Ile Asp Tyr Asp Glu His Lys Thr Met Asn Pro GluGln Ala Arg Gly 305 310 315 320 gtt ctc aag tgt gac att aca gat ctc caaatc att tgc tcc ttg gga 1009 Val Leu Lys Cys Asp Ile Thr Asp Leu Gln IleIle Cys Ser Leu Gly 325 330 335 tat agc att ctt ctc atg gtc aca tgt actgtg tat gcc atc aag act 1057 Tyr Ser Ile Leu Leu Met Val Thr Cys Thr ValTyr Ala Ile Lys Thr 340 345 350 cgg ggt gta ccc gag aat ttt aac gaa gccaag ccc att gga ttc act 1105 Arg Gly Val Pro Glu Asn Phe Asn Glu Ala LysPro Ile Gly Phe Thr 355 360 365 atg tac acg aca tgt ata gta tgg ctt gccttc att cca att ttt ttt 1153 Met Tyr Thr Thr Cys Ile Val Trp Leu Ala PheIle Pro Ile Phe Phe 370 375 380 ggc acc gct caa tca gcg gaa aag ctc tacata caa act acc acg ctt 1201 Gly Thr Ala Gln Ser Ala Glu Lys Leu Tyr IleGln Thr Thr Thr Leu 385 390 395 400 aca atc tcc atg aac cta agt gca tcagtg gcg ctg ggg atg cta tac 1249 Thr Ile Ser Met Asn Leu Ser Ala Ser ValAla Leu Gly Met Leu Tyr 405 410 415 atg ccg aaa gtg tac atc atc att ttccac cct gaa ctc aat gtc cag 1297 Met Pro Lys Val Tyr Ile Ile Ile Phe HisPro Glu Leu Asn Val Gln 420 425 430 aaa cgg aag cga agc ttc aag gcg gtagtc aca gca gcc acc atg tca 1345 Lys Arg Lys Arg Ser Phe Lys Ala Val ValThr Ala Ala Thr Met Ser 435 440 445 tcg agg ctg tca cac aaa ccc agt gacaga ccc aac ggt gag gca aag 1393 Ser Arg Leu Ser His Lys Pro Ser Asp ArgPro Asn Gly Glu Ala Lys 450 455 460 acc gag ctc tgt gaa aac gta gac ccaaac agt gag aag tgc aac tgc 1441 Thr Glu Leu Cys Glu Asn Val Asp Pro AsnSer Glu Lys Cys Asn Cys 465 470 475 480 tac tga ccatctgcac tggcatctagtcaagcgatt gtctgaggaa aggattttgg 1497 Tyr agattcccat ctgatattcttctatttggt ctcttgtacc cattgtcatc ctgtaccaca 1557 cataataaag tttaagaatgtcaagcaaaa g 1588 8 481 PRT Homo sapiens 8 Asn Lys Asp Leu Cys Ala AspTyr Arg Gly Val Cys Pro Glu Met Glu 1 5 10 15 Gln Ala Gly Gly Lys LysLeu Leu Lys Tyr Ile Arg Asn Val Asn Phe 20 25 30 Asn Gly Ser Ala Gly ThrPro Val Met Phe Asn Lys Asn Gly Asp Ala 35 40 45 Pro Gly Arg Tyr Asp IlePhe Gln Tyr Gln Thr Thr Asn Thr Ser Asn 50 55 60 Pro Gly Tyr Arg Leu IleGly Gln Trp Thr Asp Glu Leu Gln Leu Asn 65 70 75 80 Ile Glu Asp Met GlnTrp Gly Lys Gly Val Arg Glu Ile Pro Ala Ser 85 90 95 Val Cys Thr Leu ProCys Lys Pro Gly Gln Arg Lys Lys Thr Gln Lys 100 105 110 Gly Thr Pro CysCys Trp Thr Cys Glu Pro Cys Asp Gly Tyr Gln Tyr 115 120 125 Gln Phe AspGlu Met Thr Cys Gln His Cys Pro Tyr Asp Gln Arg Pro 130 135 140 Asn GluAsn Arg Thr Gly Cys Gln Asp Ile Pro Ile Ile Lys Leu Glu 145 150 155 160Trp His Ser Pro Trp Ala Val Ile Pro Val Phe Leu Ala Met Leu Gly 165 170175 Ile Ile Ala Thr Ile Phe Val Met Ala Thr Phe Ile Arg Tyr Asn Asp 180185 190 Thr Pro Ile Val Arg Ala Ser Gly Arg Glu Leu Ser Tyr Val Leu Leu195 200 205 Thr Gly Ile Phe Leu Cys Tyr Ile Ile Thr Phe Leu Met Ile AlaLys 210 215 220 Pro Asp Val Ala Val Cys Ser Phe Arg Arg Val Phe Leu GlyLeu Gly 225 230 235 240 Met Cys Ile Ser Tyr Ala Ala Leu Leu Thr Lys ThrAsn Arg Ile Tyr 245 250 255 Arg Ile Phe Glu Gln Gly Lys Lys Ser Val ThrAla Pro Arg Leu Ile 260 265 270 Ser Pro Thr Ser Gln Leu Ala Ile Thr SerSer Leu Ile Ser Val Gln 275 280 285 Leu Leu Gly Val Phe Ile Trp Phe GlyVal Asp Pro Pro Asn Ile Ile 290 295 300 Ile Asp Tyr Asp Glu His Lys ThrMet Asn Pro Glu Gln Ala Arg Gly 305 310 315 320 Val Leu Lys Cys Asp IleThr Asp Leu Gln Ile Ile Cys Ser Leu Gly 325 330 335 Tyr Ser Ile Leu LeuMet Val Thr Cys Thr Val Tyr Ala Ile Lys Thr 340 345 350 Arg Gly Val ProGlu Asn Phe Asn Glu Ala Lys Pro Ile Gly Phe Thr 355 360 365 Met Tyr ThrThr Cys Ile Val Trp Leu Ala Phe Ile Pro Ile Phe Phe 370 375 380 Gly ThrAla Gln Ser Ala Glu Lys Leu Tyr Ile Gln Thr Thr Thr Leu 385 390 395 400Thr Ile Ser Met Asn Leu Ser Ala Ser Val Ala Leu Gly Met Leu Tyr 405 410415 Met Pro Lys Val Tyr Ile Ile Ile Phe His Pro Glu Leu Asn Val Gln 420425 430 Lys Arg Lys Arg Ser Phe Lys Ala Val Val Thr Ala Ala Thr Met Ser435 440 445 Ser Arg Leu Ser His Lys Pro Ser Asp Arg Pro Asn Gly Glu AlaLys 450 455 460 Thr Glu Leu Cys Glu Asn Val Asp Pro Asn Ser Glu Lys CysAsn Cys 465 470 475 480 Tyr 9 558 DNA Homo sapiens CDS (1)..(558) 9 atggtc cag ctg agg aag ctg ctc cgc gtc ctg act ttg atg aag ttc 48 Met ValGln Leu Arg Lys Leu Leu Arg Val Leu Thr Leu Met Lys Phe 1 5 10 15 ccctgc tgc gtg ctg gag gtg ctc ctg tgc gcg ctg gcg gcg gcg gcg 96 Pro CysCys Val Leu Glu Val Leu Leu Cys Ala Leu Ala Ala Ala Ala 20 25 30 cgc ggccag gag atg tac gcc ccg cac tca atc cgg atc gag ggg gac 144 Arg Gly GlnGlu Met Tyr Ala Pro His Ser Ile Arg Ile Glu Gly Asp 35 40 45 gtc acc ctcggg ggg ctg ttc ccc gta cac gcc aag ggt ccc agc gga 192 Val Thr Leu GlyGly Leu Phe Pro Val His Ala Lys Gly Pro Ser Gly 50 55 60 gtg ccc tgc ggcgac atc aag agg gaa aac ggg atc cac agg ctg gaa 240 Val Pro Cys Gly AspIle Lys Arg Glu Asn Gly Ile His Arg Leu Glu 65 70 75 80 gcg atg ctc tacgcc ctg gac cag atc aac agt gat ccc aac cta ctg 288 Ala Met Leu Tyr AlaLeu Asp Gln Ile Asn Ser Asp Pro Asn Leu Leu 85 90 95 ccc aac gtg acg ctgggc gcg cgg atc ctg gac act tgt tcc agg gac 336 Pro Asn Val Thr Leu GlyAla Arg Ile Leu Asp Thr Cys Ser Arg Asp 100 105 110 act tac gcg ctc gaacag tcg ctt act ttc gtc cag gcg ctc atc cag 384 Thr Tyr Ala Leu Glu GlnSer Leu Thr Phe Val Gln Ala Leu Ile Gln 115 120 125 aag gac acc tcc gacgtg cgc tgc acc aac ggc gaa ccg ccg gtt ttc 432 Lys Asp Thr Ser Asp ValArg Cys Thr Asn Gly Glu Pro Pro Val Phe 130 135 140 gtc aag ccg gag aaagta gtt gga gtg att ggg gct tcg ggg agt tcg 480 Val Lys Pro Glu Lys ValVal Gly Val Ile Gly Ala Ser Gly Ser Ser 145 150 155 160 gtc tcc atc atggta gcc aac atc ctg agg ctc ttc cag atc ccc cag 528 Val Ser Ile Met ValAla Asn Ile Leu Arg Leu Phe Gln Ile Pro Gln 165 170 175 att agt tat gcatca acg gca ccc gag cta 558 Ile Ser Tyr Ala Ser Thr Ala Pro Glu Leu 180185 10 186 PRT Homo sapiens 10 Met Val Gln Leu Arg Lys Leu Leu Arg ValLeu Thr Leu Met Lys Phe 1 5 10 15 Pro Cys Cys Val Leu Glu Val Leu LeuCys Ala Leu Ala Ala Ala Ala 20 25 30 Arg Gly Gln Glu Met Tyr Ala Pro HisSer Ile Arg Ile Glu Gly Asp 35 40 45 Val Thr Leu Gly Gly Leu Phe Pro ValHis Ala Lys Gly Pro Ser Gly 50 55 60 Val Pro Cys Gly Asp Ile Lys Arg GluAsn Gly Ile His Arg Leu Glu 65 70 75 80 Ala Met Leu Tyr Ala Leu Asp GlnIle Asn Ser Asp Pro Asn Leu Leu 85 90 95 Pro Asn Val Thr Leu Gly Ala ArgIle Leu Asp Thr Cys Ser Arg Asp 100 105 110 Thr Tyr Ala Leu Glu Gln SerLeu Thr Phe Val Gln Ala Leu Ile Gln 115 120 125 Lys Asp Thr Ser Asp ValArg Cys Thr Asn Gly Glu Pro Pro Val Phe 130 135 140 Val Lys Pro Glu LysVal Val Gly Val Ile Gly Ala Ser Gly Ser Ser 145 150 155 160 Val Ser IleMet Val Ala Asn Ile Leu Arg Leu Phe Gln Ile Pro Gln 165 170 175 Ile SerTyr Ala Ser Thr Ala Pro Glu Leu 180 185 11 2745 DNA Homo sapiens CDS(1)..(2745) 11 atg gtc cag ctg agg aag ctg ctc cgc gtc ctg act ttg atgaag ttc 48 Met Val Gln Leu Arg Lys Leu Leu Arg Val Leu Thr Leu Met LysPhe 1 5 10 15 ccc tgc tgc gtg ctg gag gtg ctc ctg tgc gcg ctg gcg gcggcg gcg 96 Pro Cys Cys Val Leu Glu Val Leu Leu Cys Ala Leu Ala Ala AlaAla 20 25 30 cgc ggc cag gag atg tac gcc ccg cac tca atc cgg atc gag ggggac 144 Arg Gly Gln Glu Met Tyr Ala Pro His Ser Ile Arg Ile Glu Gly Asp35 40 45 gtc acc ctc ggg ggg ctg ttc ccc gta cac gcc aag ggt ccc agc gga192 Val Thr Leu Gly Gly Leu Phe Pro Val His Ala Lys Gly Pro Ser Gly 5055 60 gtg ccc tgc ggc gac atc aag agg gaa aac ggg atc cac agg ctg gaa240 Val Pro Cys Gly Asp Ile Lys Arg Glu Asn Gly Ile His Arg Leu Glu 6570 75 80 gcg atg ctc tac gcc ctg gac cag atc aac agt gat ccc aac cta ctg288 Ala Met Leu Tyr Ala Leu Asp Gln Ile Asn Ser Asp Pro Asn Leu Leu 8590 95 ccc aac gtg acg ctg ggc gcg cgg atc ctg gac act tgt tcc agg gac336 Pro Asn Val Thr Leu Gly Ala Arg Ile Leu Asp Thr Cys Ser Arg Asp 100105 110 act tac gcg ctc gaa cag tcg ctt act ttc gtc cag gcg ctc atc cag384 Thr Tyr Ala Leu Glu Gln Ser Leu Thr Phe Val Gln Ala Leu Ile Gln 115120 125 aag gac acc tcc gac gtg cgc tgc acc aac ggc gaa ccg ccg gtt ttc432 Lys Asp Thr Ser Asp Val Arg Cys Thr Asn Gly Glu Pro Pro Val Phe 130135 140 gtc aag ccg gag aaa gta gtt gga gtg att ggg gct tcg ggg agt tcg480 Val Lys Pro Glu Lys Val Val Gly Val Ile Gly Ala Ser Gly Ser Ser 145150 155 160 gtc tcc atc atg gta gcc aac atc ctg agg ctc ttc cag atc ccccag 528 Val Ser Ile Met Val Ala Asn Ile Leu Arg Leu Phe Gln Ile Pro Gln165 170 175 att agt tat gca tca acg gca ccc gag cta agt gat gac cgg cgctat 576 Ile Ser Tyr Ala Ser Thr Ala Pro Glu Leu Ser Asp Asp Arg Arg Tyr180 185 190 gac ttc ttc tct cgc gtg gtg cca ccc gat tcc ttc caa gcc caggcc 624 Asp Phe Phe Ser Arg Val Val Pro Pro Asp Ser Phe Gln Ala Gln Ala195 200 205 atg gta gac att gta aag gcc cta ggc tgg aat tat gtg tct accctc 672 Met Val Asp Ile Val Lys Ala Leu Gly Trp Asn Tyr Val Ser Thr Leu210 215 220 gca tcg gaa gga agt tat gga gag aaa ggt gtg gag tcc ttc acgcag 720 Ala Ser Glu Gly Ser Tyr Gly Glu Lys Gly Val Glu Ser Phe Thr Gln225 230 235 240 att tcc aaa gag gca ggt gga ctc tgc att gcc cag tcc gtgaga atc 768 Ile Ser Lys Glu Ala Gly Gly Leu Cys Ile Ala Gln Ser Val ArgIle 245 250 255 ccc cag gaa cgc aaa gac agg acc att gac ttt gat aga attatc aaa 816 Pro Gln Glu Arg Lys Asp Arg Thr Ile Asp Phe Asp Arg Ile IleLys 260 265 270 cag ctc ctg gac acc ccc aac tcc agg gcc gtc gtg att tttgcc aac 864 Gln Leu Leu Asp Thr Pro Asn Ser Arg Ala Val Val Ile Phe AlaAsn 275 280 285 gat gag gat ata aag cag atc ctt gca gca gcc aaa aga gctgac caa 912 Asp Glu Asp Ile Lys Gln Ile Leu Ala Ala Ala Lys Arg Ala AspGln 290 295 300 gtt ggc cat ttt ctt tgg gtg gga tca gac agc tgg gga tccaaa ata 960 Val Gly His Phe Leu Trp Val Gly Ser Asp Ser Trp Gly Ser LysIle 305 310 315 320 aac cca ctg cac cag cat gaa gat atc gca gaa ggg gccatc acc att 1008 Asn Pro Leu His Gln His Glu Asp Ile Ala Glu Gly Ala IleThr Ile 325 330 335 cag ccc aag cga gcc acg gtg gaa ggg ttt gat gcc tacttt acg tcc 1056 Gln Pro Lys Arg Ala Thr Val Glu Gly Phe Asp Ala Tyr PheThr Ser 340 345 350 cgt aca ctt gaa aac aac aga aga aat gta tgg ttt gccgaa tac tgg 1104 Arg Thr Leu Glu Asn Asn Arg Arg Asn Val Trp Phe Ala GluTyr Trp 355 360 365 gag gaa aac ttc aac tgc aag ttg acg att agt ggg tcaaaa aaa gaa 1152 Glu Glu Asn Phe Asn Cys Lys Leu Thr Ile Ser Gly Ser LysLys Glu 370 375 380 gac aca gat cgc aaa tgc aca gga cag gag aga att ggaaaa gat tcc 1200 Asp Thr Asp Arg Lys Cys Thr Gly Gln Glu Arg Ile Gly LysAsp Ser 385 390 395 400 aac tat gag cag gag ggt aaa gtc cag ttc gtg attgac gca gtc tat 1248 Asn Tyr Glu Gln Glu Gly Lys Val Gln Phe Val Ile AspAla Val Tyr 405 410 415 gct atg gct cac gcc ctt cac cac atg aac aag gatctc tgt gct gac 1296 Ala Met Ala His Ala Leu His His Met Asn Lys Asp LeuCys Ala Asp 420 425 430 tac cgg ggt gtc tgc cca gag atg gag caa gct ggaggc aag aag ttg 1344 Tyr Arg Gly Val Cys Pro Glu Met Glu Gln Ala Gly GlyLys Lys Leu 435 440 445 ctg aag tat ata cgc aat gtt aat ttc aat ggt agtgct ggc act cca 1392 Leu Lys Tyr Ile Arg Asn Val Asn Phe Asn Gly Ser AlaGly Thr Pro 450 455 460 gtg atg ttt aac aag aac ggg gat gca cct ggg cgttat gac atc ttt 1440 Val Met Phe Asn Lys Asn Gly Asp Ala Pro Gly Arg TyrAsp Ile Phe 465 470 475 480 cag tac cag acc aca aac acc agc aac ccg ggttac cgt ctg atc ggg 1488 Gln Tyr Gln Thr Thr Asn Thr Ser Asn Pro Gly TyrArg Leu Ile Gly 485 490 495 cag tgg aca gac gaa ctt cag ctc aat ata gaagac atg cag tgg ggt 1536 Gln Trp Thr Asp Glu Leu Gln Leu Asn Ile Glu AspMet Gln Trp Gly 500 505 510 aaa gga gtc cga gag ata ccc gcc tca gtg tgcaca cta cca tgt aag 1584 Lys Gly Val Arg Glu Ile Pro Ala Ser Val Cys ThrLeu Pro Cys Lys 515 520 525 cca gga cag aga aag aag aca cag aaa gga actcct tgc tgt tgg acc 1632 Pro Gly Gln Arg Lys Lys Thr Gln Lys Gly Thr ProCys Cys Trp Thr 530 535 540 tgt gag cct tgc gat ggt tac cag tac cag tttgat gag atg aca tgc 1680 Cys Glu Pro Cys Asp Gly Tyr Gln Tyr Gln Phe AspGlu Met Thr Cys 545 550 555 560 cag cat tgc ccc tat gac cag agg ccc aatgaa aat cga acc gga tgc 1728 Gln His Cys Pro Tyr Asp Gln Arg Pro Asn GluAsn Arg Thr Gly Cys 565 570 575 cag gat att ccc atc atc aaa ctg gag tggcac tcc ccc tgg gct gtg 1776 Gln Asp Ile Pro Ile Ile Lys Leu Glu Trp HisSer Pro Trp Ala Val 580 585 590 att cct gtc ttc ctg gca atg ttg ggg atcatt gcc acc atc ttt gtc 1824 Ile Pro Val Phe Leu Ala Met Leu Gly Ile IleAla Thr Ile Phe Val 595 600 605 atg gcc act ttc atc cgc tac aat gac acgccc att gtc cgg gca tct 1872 Met Ala Thr Phe Ile Arg Tyr Asn Asp Thr ProIle Val Arg Ala Ser 610 615 620 ggg cgg gaa ctc agc tat gtt ctt ttg acgggc atc ttt ctt tgc tac 1920 Gly Arg Glu Leu Ser Tyr Val Leu Leu Thr GlyIle Phe Leu Cys Tyr 625 630 635 640 atc atc act ttc ctg atg att gcc aaacca gat gtg gca gtg tgt tct 1968 Ile Ile Thr Phe Leu Met Ile Ala Lys ProAsp Val Ala Val Cys Ser 645 650 655 ttc cgg cga gtt ttc ttg ggc ttg ggtatg tgc atc agt tat gca gcc 2016 Phe Arg Arg Val Phe Leu Gly Leu Gly MetCys Ile Ser Tyr Ala Ala 660 665 670 ctc ttg acg aaa aca aat cgg att tatcgc ata ttt gag cag ggc aag 2064 Leu Leu Thr Lys Thr Asn Arg Ile Tyr ArgIle Phe Glu Gln Gly Lys 675 680 685 aaa tca gta aca gct ccc aga ctc ataagc cca aca tca caa ctg gca 2112 Lys Ser Val Thr Ala Pro Arg Leu Ile SerPro Thr Ser Gln Leu Ala 690 695 700 atc act tcc agt tta ata tca gtt cagctt cta ggg gtg ttc att tgg 2160 Ile Thr Ser Ser Leu Ile Ser Val Gln LeuLeu Gly Val Phe Ile Trp 705 710 715 720 ttt ggt gtt gat cca ccc aac atcatc ata gac tac gat gaa cac aag 2208 Phe Gly Val Asp Pro Pro Asn Ile IleIle Asp Tyr Asp Glu His Lys 725 730 735 aca atg aac cct gag caa gcc agaggg gtt ctc aag tgt gac att aca 2256 Thr Met Asn Pro Glu Gln Ala Arg GlyVal Leu Lys Cys Asp Ile Thr 740 745 750 gat ctc caa atc att tgc tcc ttggga tat agc att ctt ctc atg gtc 2304 Asp Leu Gln Ile Ile Cys Ser Leu GlyTyr Ser Ile Leu Leu Met Val 755 760 765 aca tgt act gtg tat gcc atc aagact cgg ggt gta ccc gag aat ttt 2352 Thr Cys Thr Val Tyr Ala Ile Lys ThrArg Gly Val Pro Glu Asn Phe 770 775 780 aac gaa gcc aag ccc att gga ttcact atg tac acg aca tgt ata gta 2400 Asn Glu Ala Lys Pro Ile Gly Phe ThrMet Tyr Thr Thr Cys Ile Val 785 790 795 800 tgg ctt gcc ttc att cca attttt ttt ggc acc gct caa tca gcg gaa 2448 Trp Leu Ala Phe Ile Pro Ile PhePhe Gly Thr Ala Gln Ser Ala Glu 805 810 815 aag ctc tac ata caa act accacg ctt aca atc tcc atg aac cta agt 2496 Lys Leu Tyr Ile Gln Thr Thr ThrLeu Thr Ile Ser Met Asn Leu Ser 820 825 830 gca tca gtg gcg ctg ggg atgcta tac atg ccg aaa gtg tac atc atc 2544 Ala Ser Val Ala Leu Gly Met LeuTyr Met Pro Lys Val Tyr Ile Ile 835 840 845 att ttc cac cct gaa ctc aatgtc cag aaa cgg aag cga agc ttc aag 2592 Ile Phe His Pro Glu Leu Asn ValGln Lys Arg Lys Arg Ser Phe Lys 850 855 860 gcg gta gtc aca gca gcc accatg tca tcg agg ctg tca cac aaa ccc 2640 Ala Val Val Thr Ala Ala Thr MetSer Ser Arg Leu Ser His Lys Pro 865 870 875 880 agt gac aga ccc aac ggtgag gca aag acc gag ctc tgt gaa aac gta 2688 Ser Asp Arg Pro Asn Gly GluAla Lys Thr Glu Leu Cys Glu Asn Val 885 890 895 gac cca aac agc cct gctgca aaa aag aag tat gtc agt tat aat aac 2736 Asp Pro Asn Ser Pro Ala AlaLys Lys Lys Tyr Val Ser Tyr Asn Asn 900 905 910 ctg gtt atc 2745 Leu ValIle 915 12 915 PRT Homo sapiens 12 Met Val Gln Leu Arg Lys Leu Leu ArgVal Leu Thr Leu Met Lys Phe 1 5 10 15 Pro Cys Cys Val Leu Glu Val LeuLeu Cys Ala Leu Ala Ala Ala Ala 20 25 30 Arg Gly Gln Glu Met Tyr Ala ProHis Ser Ile Arg Ile Glu Gly Asp 35 40 45 Val Thr Leu Gly Gly Leu Phe ProVal His Ala Lys Gly Pro Ser Gly 50 55 60 Val Pro Cys Gly Asp Ile Lys ArgGlu Asn Gly Ile His Arg Leu Glu 65 70 75 80 Ala Met Leu Tyr Ala Leu AspGln Ile Asn Ser Asp Pro Asn Leu Leu 85 90 95 Pro Asn Val Thr Leu Gly AlaArg Ile Leu Asp Thr Cys Ser Arg Asp 100 105 110 Thr Tyr Ala Leu Glu GlnSer Leu Thr Phe Val Gln Ala Leu Ile Gln 115 120 125 Lys Asp Thr Ser AspVal Arg Cys Thr Asn Gly Glu Pro Pro Val Phe 130 135 140 Val Lys Pro GluLys Val Val Gly Val Ile Gly Ala Ser Gly Ser Ser 145 150 155 160 Val SerIle Met Val Ala Asn Ile Leu Arg Leu Phe Gln Ile Pro Gln 165 170 175 IleSer Tyr Ala Ser Thr Ala Pro Glu Leu Ser Asp Asp Arg Arg Tyr 180 185 190Asp Phe Phe Ser Arg Val Val Pro Pro Asp Ser Phe Gln Ala Gln Ala 195 200205 Met Val Asp Ile Val Lys Ala Leu Gly Trp Asn Tyr Val Ser Thr Leu 210215 220 Ala Ser Glu Gly Ser Tyr Gly Glu Lys Gly Val Glu Ser Phe Thr Gln225 230 235 240 Ile Ser Lys Glu Ala Gly Gly Leu Cys Ile Ala Gln Ser ValArg Ile 245 250 255 Pro Gln Glu Arg Lys Asp Arg Thr Ile Asp Phe Asp ArgIle Ile Lys 260 265 270 Gln Leu Leu Asp Thr Pro Asn Ser Arg Ala Val ValIle Phe Ala Asn 275 280 285 Asp Glu Asp Ile Lys Gln Ile Leu Ala Ala AlaLys Arg Ala Asp Gln 290 295 300 Val Gly His Phe Leu Trp Val Gly Ser AspSer Trp Gly Ser Lys Ile 305 310 315 320 Asn Pro Leu His Gln His Glu AspIle Ala Glu Gly Ala Ile Thr Ile 325 330 335 Gln Pro Lys Arg Ala Thr ValGlu Gly Phe Asp Ala Tyr Phe Thr Ser 340 345 350 Arg Thr Leu Glu Asn AsnArg Arg Asn Val Trp Phe Ala Glu Tyr Trp 355 360 365 Glu Glu Asn Phe AsnCys Lys Leu Thr Ile Ser Gly Ser Lys Lys Glu 370 375 380 Asp Thr Asp ArgLys Cys Thr Gly Gln Glu Arg Ile Gly Lys Asp Ser 385 390 395 400 Asn TyrGlu Gln Glu Gly Lys Val Gln Phe Val Ile Asp Ala Val Tyr 405 410 415 AlaMet Ala His Ala Leu His His Met Asn Lys Asp Leu Cys Ala Asp 420 425 430Tyr Arg Gly Val Cys Pro Glu Met Glu Gln Ala Gly Gly Lys Lys Leu 435 440445 Leu Lys Tyr Ile Arg Asn Val Asn Phe Asn Gly Ser Ala Gly Thr Pro 450455 460 Val Met Phe Asn Lys Asn Gly Asp Ala Pro Gly Arg Tyr Asp Ile Phe465 470 475 480 Gln Tyr Gln Thr Thr Asn Thr Ser Asn Pro Gly Tyr Arg LeuIle Gly 485 490 495 Gln Trp Thr Asp Glu Leu Gln Leu Asn Ile Glu Asp MetGln Trp Gly 500 505 510 Lys Gly Val Arg Glu Ile Pro Ala Ser Val Cys ThrLeu Pro Cys Lys 515 520 525 Pro Gly Gln Arg Lys Lys Thr Gln Lys Gly ThrPro Cys Cys Trp Thr 530 535 540 Cys Glu Pro Cys Asp Gly Tyr Gln Tyr GlnPhe Asp Glu Met Thr Cys 545 550 555 560 Gln His Cys Pro Tyr Asp Gln ArgPro Asn Glu Asn Arg Thr Gly Cys 565 570 575 Gln Asp Ile Pro Ile Ile LysLeu Glu Trp His Ser Pro Trp Ala Val 580 585 590 Ile Pro Val Phe Leu AlaMet Leu Gly Ile Ile Ala Thr Ile Phe Val 595 600 605 Met Ala Thr Phe IleArg Tyr Asn Asp Thr Pro Ile Val Arg Ala Ser 610 615 620 Gly Arg Glu LeuSer Tyr Val Leu Leu Thr Gly Ile Phe Leu Cys Tyr 625 630 635 640 Ile IleThr Phe Leu Met Ile Ala Lys Pro Asp Val Ala Val Cys Ser 645 650 655 PheArg Arg Val Phe Leu Gly Leu Gly Met Cys Ile Ser Tyr Ala Ala 660 665 670Leu Leu Thr Lys Thr Asn Arg Ile Tyr Arg Ile Phe Glu Gln Gly Lys 675 680685 Lys Ser Val Thr Ala Pro Arg Leu Ile Ser Pro Thr Ser Gln Leu Ala 690695 700 Ile Thr Ser Ser Leu Ile Ser Val Gln Leu Leu Gly Val Phe Ile Trp705 710 715 720 Phe Gly Val Asp Pro Pro Asn Ile Ile Ile Asp Tyr Asp GluHis Lys 725 730 735 Thr Met Asn Pro Glu Gln Ala Arg Gly Val Leu Lys CysAsp Ile Thr 740 745 750 Asp Leu Gln Ile Ile Cys Ser Leu Gly Tyr Ser IleLeu Leu Met Val 755 760 765 Thr Cys Thr Val Tyr Ala Ile Lys Thr Arg GlyVal Pro Glu Asn Phe 770 775 780 Asn Glu Ala Lys Pro Ile Gly Phe Thr MetTyr Thr Thr Cys Ile Val 785 790 795 800 Trp Leu Ala Phe Ile Pro Ile PhePhe Gly Thr Ala Gln Ser Ala Glu 805 810 815 Lys Leu Tyr Ile Gln Thr ThrThr Leu Thr Ile Ser Met Asn Leu Ser 820 825 830 Ala Ser Val Ala Leu GlyMet Leu Tyr Met Pro Lys Val Tyr Ile Ile 835 840 845 Ile Phe His Pro GluLeu Asn Val Gln Lys Arg Lys Arg Ser Phe Lys 850 855 860 Ala Val Val ThrAla Ala Thr Met Ser Ser Arg Leu Ser His Lys Pro 865 870 875 880 Ser AspArg Pro Asn Gly Glu Ala Lys Thr Glu Leu Cys Glu Asn Val 885 890 895 AspPro Asn Ser Pro Ala Ala Lys Lys Lys Tyr Val Ser Tyr Asn Asn 900 905 910Leu Val Ile 915 13 2766 DNA Homo sapiens CDS (1)..(2766) 13 atg gtc cagctg agg aag ctg ctc cgc gtc ctg act ttg atg aag ttc 48 Met Val Gln LeuArg Lys Leu Leu Arg Val Leu Thr Leu Met Lys Phe 1 5 10 15 ccc tgc tgcgtg ctg gag gtg ctc ctg tgc gcg ctg gcg gcg gcg gcg 96 Pro Cys Cys ValLeu Glu Val Leu Leu Cys Ala Leu Ala Ala Ala Ala 20 25 30 cgc ggc cag gagatg tac gcc ccg cac tca atc cgg atc gag ggg gac 144 Arg Gly Gln Glu MetTyr Ala Pro His Ser Ile Arg Ile Glu Gly Asp 35 40 45 gtc acc ctc ggg gggctg ttc ccc gta cac gcc aag ggt ccc agc gga 192 Val Thr Leu Gly Gly LeuPhe Pro Val His Ala Lys Gly Pro Ser Gly 50 55 60 gtg ccc tgc ggc gac atcaag agg gaa aac ggg atc cac agg ctg gaa 240 Val Pro Cys Gly Asp Ile LysArg Glu Asn Gly Ile His Arg Leu Glu 65 70 75 80 gcg atg ctc tac gcc ctggac cag atc aac agt gat ccc aac cta ctg 288 Ala Met Leu Tyr Ala Leu AspGln Ile Asn Ser Asp Pro Asn Leu Leu 85 90 95 ccc aac gtg acg ctg ggc gcgcgg atc ctg gac act tgt tcc agg gac 336 Pro Asn Val Thr Leu Gly Ala ArgIle Leu Asp Thr Cys Ser Arg Asp 100 105 110 act tac gcg ctc gaa cag tcgctt act ttc gtc cag gcg ctc atc cag 384 Thr Tyr Ala Leu Glu Gln Ser LeuThr Phe Val Gln Ala Leu Ile Gln 115 120 125 aag gac acc tcc gac gtg cgctgc acc aac ggc gaa ccg ccg gtt ttc 432 Lys Asp Thr Ser Asp Val Arg CysThr Asn Gly Glu Pro Pro Val Phe 130 135 140 gtc aag ccg gag aaa gta gttgga gtg att ggg gct tcg ggg agt tcg 480 Val Lys Pro Glu Lys Val Val GlyVal Ile Gly Ala Ser Gly Ser Ser 145 150 155 160 gtc tcc atc atg gta gccaac atc ctg agg ctc ttc cag atc ccc cag 528 Val Ser Ile Met Val Ala AsnIle Leu Arg Leu Phe Gln Ile Pro Gln 165 170 175 att agt tat gca tca acggca ccc gag cta agt gat gac cgg cgc tat 576 Ile Ser Tyr Ala Ser Thr AlaPro Glu Leu Ser Asp Asp Arg Arg Tyr 180 185 190 gac ttc ttc tct cgc gtggtg cca ccc gat tcc ttc caa gcc cag gcc 624 Asp Phe Phe Ser Arg Val ValPro Pro Asp Ser Phe Gln Ala Gln Ala 195 200 205 atg gta gac att gta aaggcc cta ggc tgg aat tat gtg tct acc ctc 672 Met Val Asp Ile Val Lys AlaLeu Gly Trp Asn Tyr Val Ser Thr Leu 210 215 220 gca tcg gaa gga agt tatgga gag aaa ggt gtg gag tcc ttc acg cag 720 Ala Ser Glu Gly Ser Tyr GlyGlu Lys Gly Val Glu Ser Phe Thr Gln 225 230 235 240 att tcc aaa gag gcaggt gga ctc tgc att gcc cag tcc gtg aga atc 768 Ile Ser Lys Glu Ala GlyGly Leu Cys Ile Ala Gln Ser Val Arg Ile 245 250 255 ccc cag gaa cgc aaagac agg acc att gac ttt gat aga att atc aaa 816 Pro Gln Glu Arg Lys AspArg Thr Ile Asp Phe Asp Arg Ile Ile Lys 260 265 270 cag ctc ctg gac accccc aac tcc agg gcc gtc gtg att ttt gcc aac 864 Gln Leu Leu Asp Thr ProAsn Ser Arg Ala Val Val Ile Phe Ala Asn 275 280 285 gat gag gat ata aagcag atc ctt gca gca gcc aaa aga gct gac caa 912 Asp Glu Asp Ile Lys GlnIle Leu Ala Ala Ala Lys Arg Ala Asp Gln 290 295 300 gtt ggc cat ttt ctttgg gtg gga tca gac agc tgg gga tcc aaa ata 960 Val Gly His Phe Leu TrpVal Gly Ser Asp Ser Trp Gly Ser Lys Ile 305 310 315 320 aac cca ctg caccag cat gaa gat atc gca gaa ggg gcc atc acc att 1008 Asn Pro Leu His GlnHis Glu Asp Ile Ala Glu Gly Ala Ile Thr Ile 325 330 335 cag ccc aag cgagcc acg gtg gaa ggg ttt gat gcc tac ttt acg tcc 1056 Gln Pro Lys Arg AlaThr Val Glu Gly Phe Asp Ala Tyr Phe Thr Ser 340 345 350 cgt aca ctt gaaaac aac aga aga aat gta tgg ttt gcc gaa tac tgg 1104 Arg Thr Leu Glu AsnAsn Arg Arg Asn Val Trp Phe Ala Glu Tyr Trp 355 360 365 gag gaa aac ttcaac tgc aag ttg acg att agt ggg tca aaa aaa gaa 1152 Glu Glu Asn Phe AsnCys Lys Leu Thr Ile Ser Gly Ser Lys Lys Glu 370 375 380 gac aca gat cgcaaa tgc aca gga cag gag aga att gga aaa gat tcc 1200 Asp Thr Asp Arg LysCys Thr Gly Gln Glu Arg Ile Gly Lys Asp Ser 385 390 395 400 aac tat gagcag gag ggt aaa gtc cag ttc gtg att gac gca gtc tat 1248 Asn Tyr Glu GlnGlu Gly Lys Val Gln Phe Val Ile Asp Ala Val Tyr 405 410 415 gct atg gctcac gcc ctt cac cac atg aac aag gat ctc tgt gct gac 1296 Ala Met Ala HisAla Leu His His Met Asn Lys Asp Leu Cys Ala Asp 420 425 430 tac cgg ggtgtc tgc cca gag atg gag caa gct gga ggc aag aag ttg 1344 Tyr Arg Gly ValCys Pro Glu Met Glu Gln Ala Gly Gly Lys Lys Leu 435 440 445 ctg aag tatata cgc aat gtt aat ttc aat ggt agt gct ggc act cca 1392 Leu Lys Tyr IleArg Asn Val Asn Phe Asn Gly Ser Ala Gly Thr Pro 450 455 460 gtg atg tttaac aag aac ggg gat gca cct ggg cgt tat gac atc ttt 1440 Val Met Phe AsnLys Asn Gly Asp Ala Pro Gly Arg Tyr Asp Ile Phe 465 470 475 480 cag taccag acc aca aac acc agc aac ccg ggt tac cgt ctg atc ggg 1488 Gln Tyr GlnThr Thr Asn Thr Ser Asn Pro Gly Tyr Arg Leu Ile Gly 485 490 495 cag tggaca gac gaa ctt cag ctc aat ata gaa gac atg cag tgg ggt 1536 Gln Trp ThrAsp Glu Leu Gln Leu Asn Ile Glu Asp Met Gln Trp Gly 500 505 510 aaa ggagtc cga gag ata ccc gcc tca gtg tgc aca cta cca tgt aag 1584 Lys Gly ValArg Glu Ile Pro Ala Ser Val Cys Thr Leu Pro Cys Lys 515 520 525 cca ggacag aga aag aag aca cag aaa gga act cct tgc tgt tgg acc 1632 Pro Gly GlnArg Lys Lys Thr Gln Lys Gly Thr Pro Cys Cys Trp Thr 530 535 540 tgt gagcct tgc gat ggt tac cag tac cag ttt gat gag atg aca tgc 1680 Cys Glu ProCys Asp Gly Tyr Gln Tyr Gln Phe Asp Glu Met Thr Cys 545 550 555 560 cagcat tgc ccc tat gac cag agg ccc aat gaa aat cga acc gga tgc 1728 Gln HisCys Pro Tyr Asp Gln Arg Pro Asn Glu Asn Arg Thr Gly Cys 565 570 575 caggat att ccc atc atc aaa ctg gag tgg cac tcc ccc tgg gct gtg 1776 Gln AspIle Pro Ile Ile Lys Leu Glu Trp His Ser Pro Trp Ala Val 580 585 590 attcct gtc ttc ctg gca atg ttg ggg atc att gcc acc atc ttt gtc 1824 Ile ProVal Phe Leu Ala Met Leu Gly Ile Ile Ala Thr Ile Phe Val 595 600 605 atggcc act ttc atc cgc tac aat gac acg ccc att gtc cgg gca tct 1872 Met AlaThr Phe Ile Arg Tyr Asn Asp Thr Pro Ile Val Arg Ala Ser 610 615 620 gggcgg gaa ctc agc tat gtt ctt ttg acg ggc atc ttt ctt tgc tac 1920 Gly ArgGlu Leu Ser Tyr Val Leu Leu Thr Gly Ile Phe Leu Cys Tyr 625 630 635 640atc atc act ttc ctg atg att gcc aaa cca gat gtg gca gtg tgt tct 1968 IleIle Thr Phe Leu Met Ile Ala Lys Pro Asp Val Ala Val Cys Ser 645 650 655ttc cgg cga gtt ttc ttg ggc ttg ggt atg tgc atc agt tat gca gcc 2016 PheArg Arg Val Phe Leu Gly Leu Gly Met Cys Ile Ser Tyr Ala Ala 660 665 670ctc ttg acg aaa aca aat cgg att tat cgc ata ttt gag cag ggc aag 2064 LeuLeu Thr Lys Thr Asn Arg Ile Tyr Arg Ile Phe Glu Gln Gly Lys 675 680 685aaa tca gta aca gct ccc aga ctc ata agc cca aca tca caa ctg gca 2112 LysSer Val Thr Ala Pro Arg Leu Ile Ser Pro Thr Ser Gln Leu Ala 690 695 700atc act tcc agt tta ata tca gtt cag ctt cta ggg gtg ttc att tgg 2160 IleThr Ser Ser Leu Ile Ser Val Gln Leu Leu Gly Val Phe Ile Trp 705 710 715720 ttt ggt gtt gat cca ccc aac atc atc ata gac tac gat gaa cac aag 2208Phe Gly Val Asp Pro Pro Asn Ile Ile Ile Asp Tyr Asp Glu His Lys 725 730735 aca atg aac cct gag caa gcc aga ggg gtt ctc aag tgt gac att aca 2256Thr Met Asn Pro Glu Gln Ala Arg Gly Val Leu Lys Cys Asp Ile Thr 740 745750 gat ctc caa atc att tgc tcc ttg gga tat agc att ctt ctc atg gtc 2304Asp Leu Gln Ile Ile Cys Ser Leu Gly Tyr Ser Ile Leu Leu Met Val 755 760765 aca tgt act gtg tat gcc atc aag act cgg ggt gta ccc gag aat ttt 2352Thr Cys Thr Val Tyr Ala Ile Lys Thr Arg Gly Val Pro Glu Asn Phe 770 775780 aac gaa gcc aag ccc att gga ttc act atg tac acg aca tgt ata gta 2400Asn Glu Ala Lys Pro Ile Gly Phe Thr Met Tyr Thr Thr Cys Ile Val 785 790795 800 tgg ctt gcc ttc att cca att ttt ttt ggc acc gct caa tca gcg gaa2448 Trp Leu Ala Phe Ile Pro Ile Phe Phe Gly Thr Ala Gln Ser Ala Glu 805810 815 aag ctc tac ata caa act acc acg ctt aca atc tcc atg aac cta agt2496 Lys Leu Tyr Ile Gln Thr Thr Thr Leu Thr Ile Ser Met Asn Leu Ser 820825 830 gca tca gtg gcg ctg ggg atg cta tac atg ccg aaa gtg tac atc atc2544 Ala Ser Val Ala Leu Gly Met Leu Tyr Met Pro Lys Val Tyr Ile Ile 835840 845 att ttc cac cct gaa ctc aat gtc cag aaa cgg aag cga agc ttc aag2592 Ile Phe His Pro Glu Leu Asn Val Gln Lys Arg Lys Arg Ser Phe Lys 850855 860 gcg gta gtc aca gca gcc acc atg tca tcg agg ctg tca cac aaa ccc2640 Ala Val Val Thr Ala Ala Thr Met Ser Ser Arg Leu Ser His Lys Pro 865870 875 880 agt gac aga ccc aac ggt gag gca aag acc gag ctc tgt gaa aacgta 2688 Ser Asp Arg Pro Asn Gly Glu Ala Lys Thr Glu Leu Cys Glu Asn Val885 890 895 gac cca aac aac tgt ata cca cca gta aga aag agt gta caa aagtct 2736 Asp Pro Asn Asn Cys Ile Pro Pro Val Arg Lys Ser Val Gln Lys Ser900 905 910 gtt act tgg tac act atc cca cca aca gta 2766 Val Thr Trp TyrThr Ile Pro Pro Thr Val 915 920 14 922 PRT Homo sapiens 14 Met Val GlnLeu Arg Lys Leu Leu Arg Val Leu Thr Leu Met Lys Phe 1 5 10 15 Pro CysCys Val Leu Glu Val Leu Leu Cys Ala Leu Ala Ala Ala Ala 20 25 30 Arg GlyGln Glu Met Tyr Ala Pro His Ser Ile Arg Ile Glu Gly Asp 35 40 45 Val ThrLeu Gly Gly Leu Phe Pro Val His Ala Lys Gly Pro Ser Gly 50 55 60 Val ProCys Gly Asp Ile Lys Arg Glu Asn Gly Ile His Arg Leu Glu 65 70 75 80 AlaMet Leu Tyr Ala Leu Asp Gln Ile Asn Ser Asp Pro Asn Leu Leu 85 90 95 ProAsn Val Thr Leu Gly Ala Arg Ile Leu Asp Thr Cys Ser Arg Asp 100 105 110Thr Tyr Ala Leu Glu Gln Ser Leu Thr Phe Val Gln Ala Leu Ile Gln 115 120125 Lys Asp Thr Ser Asp Val Arg Cys Thr Asn Gly Glu Pro Pro Val Phe 130135 140 Val Lys Pro Glu Lys Val Val Gly Val Ile Gly Ala Ser Gly Ser Ser145 150 155 160 Val Ser Ile Met Val Ala Asn Ile Leu Arg Leu Phe Gln IlePro Gln 165 170 175 Ile Ser Tyr Ala Ser Thr Ala Pro Glu Leu Ser Asp AspArg Arg Tyr 180 185 190 Asp Phe Phe Ser Arg Val Val Pro Pro Asp Ser PheGln Ala Gln Ala 195 200 205 Met Val Asp Ile Val Lys Ala Leu Gly Trp AsnTyr Val Ser Thr Leu 210 215 220 Ala Ser Glu Gly Ser Tyr Gly Glu Lys GlyVal Glu Ser Phe Thr Gln 225 230 235 240 Ile Ser Lys Glu Ala Gly Gly LeuCys Ile Ala Gln Ser Val Arg Ile 245 250 255 Pro Gln Glu Arg Lys Asp ArgThr Ile Asp Phe Asp Arg Ile Ile Lys 260 265 270 Gln Leu Leu Asp Thr ProAsn Ser Arg Ala Val Val Ile Phe Ala Asn 275 280 285 Asp Glu Asp Ile LysGln Ile Leu Ala Ala Ala Lys Arg Ala Asp Gln 290 295 300 Val Gly His PheLeu Trp Val Gly Ser Asp Ser Trp Gly Ser Lys Ile 305 310 315 320 Asn ProLeu His Gln His Glu Asp Ile Ala Glu Gly Ala Ile Thr Ile 325 330 335 GlnPro Lys Arg Ala Thr Val Glu Gly Phe Asp Ala Tyr Phe Thr Ser 340 345 350Arg Thr Leu Glu Asn Asn Arg Arg Asn Val Trp Phe Ala Glu Tyr Trp 355 360365 Glu Glu Asn Phe Asn Cys Lys Leu Thr Ile Ser Gly Ser Lys Lys Glu 370375 380 Asp Thr Asp Arg Lys Cys Thr Gly Gln Glu Arg Ile Gly Lys Asp Ser385 390 395 400 Asn Tyr Glu Gln Glu Gly Lys Val Gln Phe Val Ile Asp AlaVal Tyr 405 410 415 Ala Met Ala His Ala Leu His His Met Asn Lys Asp LeuCys Ala Asp 420 425 430 Tyr Arg Gly Val Cys Pro Glu Met Glu Gln Ala GlyGly Lys Lys Leu 435 440 445 Leu Lys Tyr Ile Arg Asn Val Asn Phe Asn GlySer Ala Gly Thr Pro 450 455 460 Val Met Phe Asn Lys Asn Gly Asp Ala ProGly Arg Tyr Asp Ile Phe 465 470 475 480 Gln Tyr Gln Thr Thr Asn Thr SerAsn Pro Gly Tyr Arg Leu Ile Gly 485 490 495 Gln Trp Thr Asp Glu Leu GlnLeu Asn Ile Glu Asp Met Gln Trp Gly 500 505 510 Lys Gly Val Arg Glu IlePro Ala Ser Val Cys Thr Leu Pro Cys Lys 515 520 525 Pro Gly Gln Arg LysLys Thr Gln Lys Gly Thr Pro Cys Cys Trp Thr 530 535 540 Cys Glu Pro CysAsp Gly Tyr Gln Tyr Gln Phe Asp Glu Met Thr Cys 545 550 555 560 Gln HisCys Pro Tyr Asp Gln Arg Pro Asn Glu Asn Arg Thr Gly Cys 565 570 575 GlnAsp Ile Pro Ile Ile Lys Leu Glu Trp His Ser Pro Trp Ala Val 580 585 590Ile Pro Val Phe Leu Ala Met Leu Gly Ile Ile Ala Thr Ile Phe Val 595 600605 Met Ala Thr Phe Ile Arg Tyr Asn Asp Thr Pro Ile Val Arg Ala Ser 610615 620 Gly Arg Glu Leu Ser Tyr Val Leu Leu Thr Gly Ile Phe Leu Cys Tyr625 630 635 640 Ile Ile Thr Phe Leu Met Ile Ala Lys Pro Asp Val Ala ValCys Ser 645 650 655 Phe Arg Arg Val Phe Leu Gly Leu Gly Met Cys Ile SerTyr Ala Ala 660 665 670 Leu Leu Thr Lys Thr Asn Arg Ile Tyr Arg Ile PheGlu Gln Gly Lys 675 680 685 Lys Ser Val Thr Ala Pro Arg Leu Ile Ser ProThr Ser Gln Leu Ala 690 695 700 Ile Thr Ser Ser Leu Ile Ser Val Gln LeuLeu Gly Val Phe Ile Trp 705 710 715 720 Phe Gly Val Asp Pro Pro Asn IleIle Ile Asp Tyr Asp Glu His Lys 725 730 735 Thr Met Asn Pro Glu Gln AlaArg Gly Val Leu Lys Cys Asp Ile Thr 740 745 750 Asp Leu Gln Ile Ile CysSer Leu Gly Tyr Ser Ile Leu Leu Met Val 755 760 765 Thr Cys Thr Val TyrAla Ile Lys Thr Arg Gly Val Pro Glu Asn Phe 770 775 780 Asn Glu Ala LysPro Ile Gly Phe Thr Met Tyr Thr Thr Cys Ile Val 785 790 795 800 Trp LeuAla Phe Ile Pro Ile Phe Phe Gly Thr Ala Gln Ser Ala Glu 805 810 815 LysLeu Tyr Ile Gln Thr Thr Thr Leu Thr Ile Ser Met Asn Leu Ser 820 825 830Ala Ser Val Ala Leu Gly Met Leu Tyr Met Pro Lys Val Tyr Ile Ile 835 840845 Ile Phe His Pro Glu Leu Asn Val Gln Lys Arg Lys Arg Ser Phe Lys 850855 860 Ala Val Val Thr Ala Ala Thr Met Ser Ser Arg Leu Ser His Lys Pro865 870 875 880 Ser Asp Arg Pro Asn Gly Glu Ala Lys Thr Glu Leu Cys GluAsn Val 885 890 895 Asp Pro Asn Asn Cys Ile Pro Pro Val Arg Lys Ser ValGln Lys Ser 900 905 910 Val Thr Trp Tyr Thr Ile Pro Pro Thr Val 915 92015 630 DNA Homo sapiens CDS (1)..(630) unsure (65)..(72) Applicants areunsure of the identity of amino acids 65, 70 and 72 and nucleotidesdesignated as n could be a or g or c or t/u 15 gtg gag gcc ctg cag tggtct ggc gac ccc cac gag gtg ccc tcg tct 48 Val Glu Ala Leu Gln Trp SerGly Asp Pro His Glu Val Pro Ser Ser 1 5 10 15 ctg tgc agc ctg ccc tgcggg ccg ggg gag cgg aag aag atg gtg aag 96 Leu Cys Ser Leu Pro Cys GlyPro Gly Glu Arg Lys Lys Met Val Lys 20 25 30 ggc gtc ccc tgc tgt tgg cactgc gag gcc tgt gac ggg tac cgc ttc 144 Gly Val Pro Cys Cys Trp His CysGlu Ala Cys Asp Gly Tyr Arg Phe 35 40 45 cag gtg gac gag ttc aca tgc gaggcc tgt cct ggg tac atg agg ccc 192 Gln Val Asp Glu Phe Thr Cys Glu AlaCys Pro Gly Tyr Met Arg Pro 50 55 60 acn ccc aac cac atc nna ctt nng cccaca cct gtg gtg cgc ctg agc 240 Xaa Pro Asn His Ile Xaa Leu Xaa Pro ThrPro Val Val Arg Leu Ser 65 70 75 80 tgg tcc tcc ccc tgg gca gcc ccg ccgctc ctc ctg gcc gtg ctg ggc 288 Trp Ser Ser Pro Trp Ala Ala Pro Pro LeuLeu Leu Ala Val Leu Gly 85 90 95 atc gtg gcc act acc acg gtg gtg gcc accttc gtg cgg tac aac aac 336 Ile Val Ala Thr Thr Thr Val Val Ala Thr PheVal Arg Tyr Asn Asn 100 105 110 acg ccc atc gtc cgg gcc tcg ggc cga gagctc agc tac gtc ctc ctc 384 Thr Pro Ile Val Arg Ala Ser Gly Arg Glu LeuSer Tyr Val Leu Leu 115 120 125 acc ggc atc ttc ctc atc tac gcc atc accttc ctc atg gtg gct gag 432 Thr Gly Ile Phe Leu Ile Tyr Ala Ile Thr PheLeu Met Val Ala Glu 130 135 140 cct ggg gca gcg gtc tgt gcc gcc cgc aggctc ttc ctg ggc ctg ggc 480 Pro Gly Ala Ala Val Cys Ala Ala Arg Arg LeuPhe Leu Gly Leu Gly 145 150 155 160 acg acc ctc agc tac tct gcc ctg ctcacc aag acc aac cgt atc tac 528 Thr Thr Leu Ser Tyr Ser Ala Leu Leu ThrLys Thr Asn Arg Ile Tyr 165 170 175 cgc atc ttt gag cag ggc aag cgc tcggtc aca ccc cct ccc ttc atc 576 Arg Ile Phe Glu Gln Gly Lys Arg Ser ValThr Pro Pro Pro Phe Ile 180 185 190 agc ccc acc tca cag ctg gtc atc accttc agc ctc acc tcc ctg cag 624 Ser Pro Thr Ser Gln Leu Val Ile Thr PheSer Leu Thr Ser Leu Gln 195 200 205 gtg ggg 630 Val Gly 210 16 210 PRTHomo sapiens 16 Val Glu Ala Leu Gln Trp Ser Gly Asp Pro His Glu Val ProSer Ser 1 5 10 15 Leu Cys Ser Leu Pro Cys Gly Pro Gly Glu Arg Lys LysMet Val Lys 20 25 30 Gly Val Pro Cys Cys Trp His Cys Glu Ala Cys Asp GlyTyr Arg Phe 35 40 45 Gln Val Asp Glu Phe Thr Cys Glu Ala Cys Pro Gly TyrMet Arg Pro 50 55 60 Xaa Pro Asn His Ile Xaa Leu Xaa Pro Thr Pro Val ValArg Leu Ser 65 70 75 80 Trp Ser Ser Pro Trp Ala Ala Pro Pro Leu Leu LeuAla Val Leu Gly 85 90 95 Ile Val Ala Thr Thr Thr Val Val Ala Thr Phe ValArg Tyr Asn Asn 100 105 110 Thr Pro Ile Val Arg Ala Ser Gly Arg Glu LeuSer Tyr Val Leu Leu 115 120 125 Thr Gly Ile Phe Leu Ile Tyr Ala Ile ThrPhe Leu Met Val Ala Glu 130 135 140 Pro Gly Ala Ala Val Cys Ala Ala ArgArg Leu Phe Leu Gly Leu Gly 145 150 155 160 Thr Thr Leu Ser Tyr Ser AlaLeu Leu Thr Lys Thr Asn Arg Ile Tyr 165 170 175 Arg Ile Phe Glu Gln GlyLys Arg Ser Val Thr Pro Pro Pro Phe Ile 180 185 190 Ser Pro Thr Ser GlnLeu Val Ile Thr Phe Ser Leu Thr Ser Leu Gln 195 200 205 Val Gly 210 1720 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer 17 gtcaaggcct cgggccggga 20 18 21 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide primer 18ctagatggca tggttggtgt a 21 19 44 DNA Artificial Sequence Description ofArtificial Sequence Oligonucleotide primer 19 gcgctgcagg cggccgcagggcctgctagg gctaggagcg gggc 44 20 29 DNA Artificial Sequence Descriptionof Artificial Sequence Oligonucleotide primer 20 gcggaattcc ctccgtgccgtccttctcg 29 21 22 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer 21 tatcttgagt ggagtgacat ag 22 22 21 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer 22 actgcggacg ttcctctcag g 21 23 22 DNA Homo sapiens Descriptionof Artificial Sequence Oligonucleotide primer 23 aacctgagag gaacgtccgcag 22 24 24 DNA Homo sapiens Description of Artificial SequenceOligonucleotide primer 24 ctacagggtg gaagagcttt gctt 24 25 24 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer 25 tcaaagctgc gcatgtgccg acgg 24 26 24 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer 26 tcaatagacagtgttttggc ggtc 24

What is claimed is:
 1. A purified human metabotropic glutamate receptorcomprising the amino acid sequence set forth in SEQ ID NO:2.
 2. Anisolated composition which comprises: (a) a receptor of claim 1 and (b)one or more chemical entities, wherein said one or more chemicalentities is covalently bonded to or adsorptively associated with saidreceptor of claim
 1. 3. Process for the production of a receptoraccording to claim 1 which comprises culturing a suitable host celltransformed with a hybrid vector comprising an expression cassettecomprising a promoter and a DNA coding for a receptor according to claim1 wherein the DNA is controlled by said promoter; expressing thereceptor according to claim 1 in said host cell; and isolating thereceptor.
 4. An isolated nucleic acid comprising a nucleic acid sequencecoding for the amino acid sequence set forth in SEQ ID NO:2.
 5. Anisolated nucleic acid according to claim 4, wherein the nucleic acid isa DNA.
 6. A process for the preparation of a nucleic acid according toclaim 5 which comprises: chemically synthesizing said nucleic acid; orincorporating said nucleic acid into a hybrid vector comprising anorigin of replication and replicating said vector in a suitable hostorganism; or amplifying said nucleic acid by polymerase chain reaction;and isolating said nucleic acid.
 7. A DNA according to claim 5 which isa hybrid vector.
 8. An isolated host cell comprising the DNA of claim 5.9. An isolated eukaryotic host cell comprising the DNA of claim 5, anmRNA transcribed from the DNA of claim 5, and a protein translated fromsaid mRNA.
 10. An isolated host cell according to claim 8 which is amammalian cell.
 11. A process for the preparation of an isolated hostcell comprising a DNA encoding the receptor of claim 1, wherein saidprocess comprises transfection or transformation of the isolated hostcell with a hybrid vector comprising a DNA encoding said receptor. 12.An assay for identifying compounds which modulate the activity of ahmGluR receptor according to claim 1 comprising: contacting cellsexpressing DNA encoding said hmGluR receptor according to claim 1 with acompound to be tested for its ability to modulate the activity of saidreceptor under conditions appropriate for binding to said receptor; andsubsequently monitoring said cells for a resulting change in secondmessenger activity; wherein a difference in the second messengeractivity of the cell in the presence compared to the absence of saidcompound indicates that the compound modulates activity of the receptor.13. A method for identifying an agonist or an allosteric modulatorhaving agonistic activity of a hmGluR receptor according to claim 1comprising the steps of (a) exposing a test compound to a hmGluRreceptor of claim 1 coupled to a response pathway, under conditions andfor a time sufficient to allow (i) interaction of the test compound withthe receptor and (ii) stimulation of the activity of the responsepathway, and (b) detecting a stimulation in the activity of the responsepathway resulting from the interaction of the test compound with thehmGluR receptor, relative to the activity of the response pathway in theabsence of the test compound, wherein detection of a stimulation of theactivity of the response pathway relative to the activity of theresponse pathway in the absence of the tested compound indicates thatthe test compound is an agonist or an allosteric modulator havingagonist-like activity of the receptor.
 14. A method for identifying anantagonist or an allosteric modulator having antagonistic activity of ahmGluR receptor according to claim 1 comprising the steps of (a)exposing a test compound in the presence of a known glutamate agonist toa hmGluR receptor of claim 1 coupled to a response pathway, underconditions and for a time sufficient to allow (i) interaction of theagonist with the receptor and (ii) stimulation of the activity of theresponse pathway by the agonist, and (b) detecting an inhibition of thestimulation of the activity of the response pathway by the agonistresulting from the interaction of the test compound with the hmGluRreceptor, relative to the stimulation of the activity of the responsepathway induced by the glutamate agonist alone, wherein detection of aninhibition in the stimulation of the activity of the response pathwayindicates that the test compound is an antagonist or an allostericmodulator having antagonist-like activity of the receptor.
 15. A fusionprotein comprising a receptor according to claim
 1. 16. A purifiedprotein according to claim 1 consisting of the amino acid sequence setforth in SEQ ID NO:2.
 17. The composition of claim 2, wherein saidchemical entity is selected from the group consisting of acyl moieties,cell membranes, polypeptides, sugar molecules, alkyl groups, aminogroups, radioactive moieties, and fluorescent moieties.
 18. The nucleicacid of claim 4 wherein said nucleic acid sequence comprises SEQ IDNO:1.